Dual specific binding proteins directed against il-13 and/or il-17

ABSTRACT

Engineered multivalent and multispecific binding proteins that bind IL-13 and/or IL-17 are provided, along with methods of making and uses in the prevention, diagnosis, and/or treatment of disease.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/581,964, filed Dec. 30, 2011, the complete disclosure of which ishereby incorporated by reference.

FIELD

Multivalent and multispecific binding proteins that bind IL-13 and/orIL-17, methods of making, and their uses in the diagnosis, prevention,and/or treatment of acute and chronic inflammatory diseases, cancer, andother diseases are provided.

BACKGROUND

Engineered proteins, such as multispecific binding proteins capable ofbinding two or more antigens, are known in the art. Such multispecificbinding proteins can be generated using cell fusion, chemicalconjugation, or recombinant DNA techniques. There are a variety ofmultispecific binding protein structures known in the art and manystructures and methods have distinct disadvantages.

Bispecific antibodies have been produced using quadroma technology.However, the presence of mis-paired by-products and significantlyreduced production yields with this technology means that sophisticatedpurification procedures are required. Bispecific antibodies can also beproduced by chemical conjugation of two different mAbs. However, thisapproach does not yield homogeneous preparations.

Other approaches used previously include coupling of two parentalantibodies with a hetero-bifunctional crosslinker, production of tandemsingle-chain Fv molecules, diabodies, bispecific diabodies, single-chaindiabodies, and di-diabodies. However, each of these approaches havedisadvantages. In addition, a multivalent antibody construct comprisingtwo Fab repeats in the heavy chain of an IgG and capable of binding fourantigen molecules has been described (see PCT Publication No. WO 0177342and Miller et al. (2003) J. Immunol. 170(9): 4854-61).

U.S. Pat. No. 7,612,181 provides a novel family of binding proteinscapable of binding two or more antigens with high affinity, which arecalled dual variable domain binding proteins (DVD binding protein) ordual variable domain immunoglobulins (DVD-Ig™).

While a variety of structures are provided in the art, some withadvantages and disadvantages, specific constructs are required forpreparing multivalent binding proteins with specific properties andwhich bind to specific targets. Additionally, new variable domainsequences can further improve the properties of the binding proteins.

Interleukin 13 (IL-13) is a 17-kDa glycoprotein produced by activated Tcells of the Th2 lineage. The function of IL-13 includes immunoglobulinisotype switching to IgE in human B cells and suppressing inflammatorycytokine production. IL-13 is associated primarily with the induction ofairway inflammation such as asthma. It has also been linked to otherallergic diseases, fibrotic conditions, cancer and infectious diseases.

Interleukin-17 (IL-17, also referred to as IL-17A) is a 20-30 kDhomodimeric glycoprotein secreted by activated T cells at the site ofinflammation. IL-17 acts as a proinflammatory cytokine by inducing theproduction of multiple adhesion molecules, inflammatory cytokines andchemokines in various tissues to recruit monocytes and neutrophils tothe site of inflammation. IL-17 also plays an important role in thematuration of hematopoietic progenitor cells. Inappropriate or excessiveproduction of IL-17 is associated with the pathology of various diseasesor disorders including rheumatoid arthritis, asthma, lupus, allograftrejection, other inflammatory or autoimmune diseases and cancer.

There is a need in the art for improved multivalent binding proteinscapable of binding IL-13 and/or IL-17. Novel binding proteins that bindIL-13 and IL-17 are provided herein.

SUMMARY

Binding proteins capable of binding IL-13 and/or IL-17 are provided. Inan embodiment, binding proteins capable of binding IL-13 and/or IL-17with high affinity are provided.

In one embodiment, binding proteins comprising a polypeptide chain thatbinds IL-13 and/or IL-17, wherein the polypeptide chain comprisesVD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first variable domain, VD2 is asecond variable domain, C is a constant domain, X1 represents an aminoacid or polypeptide, X2 represents an Fc region and n is 0 or 1, areprovided. In an embodiment, the VD1 and VD2 in the binding protein areheavy chain variable domains. In another embodiment, VD1 and VD2 arecapable of binding the same antigen. In another embodiment, VD1 and VD2are capable of binding different antigens. In still another embodiment,C is a heavy chain constant domain. For example, X1 is a linker with theproviso that X1 is not CH1.

In an embodiment, the binding protein disclosed herein comprises apolypeptide chain that binds IL-13 and/or IL-17, wherein the polypeptidechain comprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first heavychain variable domain, VD2 is a second heavy chain variable domain, C isa heavy chain constant domain, X1 is a linker, and X2 is an Fc region.In an embodiment, X1 is a linker with the proviso that it is not CH1.

In an embodiment, the binding protein disclosed herein comprises apolypeptide chain that binds IL-13 and/or IL-17, wherein the polypeptidechain comprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain, C isa light chain constant domain, X1 is a linker, and X2 does not comprisean Fc region. In an embodiment, X1 is a linker with the proviso that itis not CL.

In another embodiment, a binding protein that binds IL-13 and/or IL-17comprising two polypeptide chains, wherein the first polypeptide chaincomprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first heavy chainvariable domain, VD2 is a second heavy chain variable domain, C is aheavy chain constant domain, X1 is a first linker, and X2 is an Fcregion; and the second polypeptide chain comprisesVD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a second linker, and X2 does not comprise an Fcregion is provided. In some embodiments, the first and second X1 are thesame. In other embodiments, the first and second X1 are different. Insome embodiments the first X1 is not a CH1 domain and/or the second X1is not a CL domain. In one embodiment, the first X1 and the second X1are short (e.g., 6 amino acid) linkers. In another embodiment, the firstX1 and the second X1 are long (e.g., greater than 6 amino acid) linkers.In another embodiment, the first X1 is a short linker and the second X1is a long linker. In another embodiment, the first X1 is a long linkerand the second X1 is a short linker.

In an embodiment, the Dual Variable Domain (DVD) binding proteincomprises four polypeptide chains, wherein each of the first twopolypeptide chains comprises VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is afirst heavy chain variable domain, VD2 is a second heavy chain variabledomain, C is a heavy chain constant domain, X1 is a first linker, and X2is an Fc region; and each of the second two polypeptide chain comprisesVD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a second linker, and X2 does not comprise an Fcregion. Such a DVD binding protein has four antigen binding sites. Insome embodiments, the first and second X1 are the same. In otherembodiments, the first and second X1 are different. In some embodiments,the first X1 is not a CH1 domain and/or the second X1 is not a CLdomain. In another embodiment, the binding proteins disclosed herein arecapable of binding IL-13 and IL-17. Accordingly, in some embodiments,the binding proteins comprise at least two variable domain sequences(e.g., VD1 and VD2) capable of binding IL-13 and IL-17, in anyorientation. In some embodiments, VD1 and VD2 are independently chosen.Therefore, in some embodiments, VD1 and VD2 comprise the same SEQ ID NOand, in other embodiments, VD1 and VD2 comprise different SEQ ID NOS.

In an embodiment,

(a) the binding protein binds IL-13 and IL-17;

(b) VD1 or VD2 heavy chain variable domain comprises three CDRs from SEQID NO: 42, 43, 44, 45, 46, 47, 48 or 49;

(c) VD1 and VD2 heavy chain variable domains independently comprisethree CDRs from SEQ ID NO: 42, 43, 44, 45, 46, 47, 48 or 49; (d) VD1heavy chain variable domain comprises three CDRs from SEQ ID NO: 36, 38,40, 42, 43, 44, 45, 46, 47, 48 or 49, and VD2 heavy chain variabledomain comprises three CDRs from SEQ ID NO: 30, 32 or 34; or

(e) VD2 heavy chain variable domain comprises three CDRs from SEQ ID NO:36, 38, 40, 42, 43, 44, 45, 46, 47, 48 or 49, and VD1 heavy chainvariable domain comprises three CDRs from SEQ ID NO: 30, 32 or 34.

In another embodiment,

(a) VD1 or VD2 heavy chain variable domain comprises SEQ ID NO: 42, 43,44, 45, 46, 47, 48 or 49;

(b) VD1 and VD2 heavy chain variable domains independently comprise SEQID NO: 42, 43, 44, 45, 46, 47, 48 or 49; (c) VD1 heavy chain variabledomain comprises SEQ ID NO: 36, 38, 40, 42, 43, 44, 45, 46, 47, 48 or49, and VD2 heavy chain variable domain comprises SEQ ID NO: 30, 32 or34; or

(d) VD2 heavy chain variable domain comprises SEQ ID NO: 36, 38, 40, 42,43, 44, 45, 46, 47, 48 or 49, and VD1 heavy chain variable domaincomprises SEQ ID NO: 30, 32 or 34.

In an embodiment,

(a) the binding protein binds IL-13 and IL-17;

(b) VD1 or VD2 light chain variable domain comprises three CDRs from SEQID NO: 50, 51, 52, 53 or 54;

(c) VD1 and VD2 light chain variable domains independently comprisethree CDRs from SEQ ID NO: 50, 51, 52, 53 or 54;

(d) VD1 light chain variable domain comprises three CDRs from SEQ ID NO:37, 39, 41, 50, 51, 52, 53 or 54, and VD2 light chain variable domaincomprises three CDRs from SEQ ID NO: 31, 33 or 35; or

(e) VD2 light chain variable domain comprises three CDRs from SEQ ID NO:37, 39, 41, 50, 51, 52, 53 or 54, and VD1 light chain variable domaincomprises three CDRs from SEQ ID NO: 31, 33 or 35.

In another embodiment,

(a) VD1 or VD2 light chain variable domain comprises SEQ ID NO: 50, 51,52, 53 or 54;

(b) VD1 and VD2 light chain variable domains independently comprise SEQID NO: 50, 51, 52, 53 or 54;

(c) VD1 light chain variable domain comprises SEQ ID NO: 37, 39, 41, 50,51, 52, 53 or 54, and VD2 light chain variable domain comprises SEQ IDNO: 31, 33 or 35; or

(d) VD2 light chain variable domain comprises SEQ ID NO: 37, 39, 41, 50,51, 52, 53 or 54, and VD1 light chain variable domain comprises SEQ IDNO: 31, 33 or 35.

In another embodiment, the binding protein comprises a heavy chain and alight chain sequence as shown in the Table 1 herein.

A further embodiment, of any of the heavy chain, light chain, two chain,or four chain embodiments, includes at least one X1 linker comprisingAKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO:5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9), SAKTTPKLEEGEFSEARV (SEQ ID NO:10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ IDNO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15);QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP(SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20);ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS(SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQID NO: 25); or GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP(SEQ ID NO: 27); ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28); or G/Sbased sequences (e.g., G4S (SEQ ID NO: 29) and G4S repeats). In anembodiment, X2 is an Fc region. In another embodiment, X2 is a variantFc region.

In still another embodiment, the Fc region, if present in the firstpolypeptide, is a native sequence Fc region or a variant sequence Fcregion. In yet another embodiment, the Fc region is an Fc region from anIgG1, an Fc region from an IgG2, an Fc region from an IgG3, an Fc regionfrom an IgG4, an Fc region from an IgA, an Fc region from an IgM, an Fcregion from an IgE, or an Fc region from an IgD.

A method of making a binding protein that binds IL-13 and/or IL-17 isprovided. In an embodiment, the method of making a binding protein thatbinds IL-13 and/or IL-17 comprises the steps of a) obtaining a firstparent antibody, or antigen binding portion thereof, that binds IL-13;b) obtaining a second parent antibody, or antigen binding portionthereof, that binds IL-17; c) preparing construct(s) encoding any of thebinding proteins described herein; and d) expressing the polypeptidechains, such that a binding protein that binds the first and the secondantigen is generated.

In any of the embodiments herein, the VD1 heavy chain variable domain,if present, and light chain variable domain, if present, can be from afirst parent antibody or antigen binding portion thereof; the VD2 heavychain variable domain, if present, and light chain variable domain, ifpresent, can be from a second parent antibody or antigen binding portionthereof. The first and second parent antibodies can be the same ordifferent.

In one embodiment, the first parent antibody or antigen binding portionthereof, binds a first antigen, and the second parent antibody orantigen binding portion thereof, binds a second antigen. In anembodiment, the first and second antigens are the same antigen. Inanother embodiment, the parent antibodies bind different epitopes on thesame antigen. In another embodiment, the first and second antigens aredifferent antigens. In another embodiment, the first parent antibody orantigen binding portion thereof, binds the first antigen with a potencydifferent from the potency with which the second parent antibody orantigen binding portion thereof, binds the second antigen. In yetanother embodiment, the first parent antibody or antigen binding portionthereof, binds the first antigen with an affinity different from theaffinity with which the second parent antibody or antigen bindingportion thereof, binds the second antigen.

In another embodiment, the first parent antibody or antigen bindingportion thereof, and the second parent antibody or antigen bindingportion thereof, are a human antibody, CDR grafted antibody, humanizedantibody, and/or affinity matured antibody.

In another embodiment, the binding protein possesses at least onedesired property exhibited by the first parent antibody or antigenbinding portion thereof, or the second parent antibody or antigenbinding portion thereof. Alternatively, the first parent antibody orantigen binding portion thereof and the second parent antibody orantigen binding portion thereof possess at least one desired propertyexhibited by the binding protein. In an embodiment, the desired propertyis one or more antibody parameters. In another embodiment, the antibodyparameters are antigen specificity, affinity to antigen, potency,biological function, epitope recognition, stability, solubility,production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, or orthologous antigenbinding. In an embodiment, the binding protein is multivalent. Inanother embodiment, the binding protein is multispecific. Themultivalent and or multispecific binding proteins described herein havedesirable properties particularly from a therapeutic standpoint. Forinstance, the multivalent and or multispecific binding protein may (1)be internalized (and/or catabolized) faster than a bivalent antibody bya cell expressing an antigen to which the antibodies bind; (2) be anagonist binding protein; and/or (3) induce cell death and/or apoptosisof a cell expressing an antigen to which the multivalent binding proteinis capable of binding. The “parent antibody”, which provides at leastone antigen binding specificity of the multivalent and or multispecificbinding protein, may be one that is internalized (and/or catabolized) bya cell expressing an antigen to which the antibody binds; and/or may bean agonist, cell death-inducing, and/or apoptosis-inducing antibody, andthe multivalent and or multispecific binding protein as described hereinmay display improvement(s) in one or more of these properties. Moreover,the parent antibody may lack any one or more of these properties, butmay acquire one or more of them when constructed as a multivalentbinding protein as described herein.

In another embodiment, the binding protein has an on rate constant(K_(on)) to one or more targets of at least about 10²M⁻¹s⁻¹; at leastabout 10³M⁻¹s⁻¹; at least about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; orat least about 10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance. Inan embodiment, the binding protein has an on rate constant (K_(on)) toone or more targets from about 10²M⁻¹s⁻¹ to about 10³M⁻¹s⁻¹; from about10³M⁻¹s⁻¹ to about 10⁴M⁻¹s⁻¹; from about 10⁴M⁻¹s⁻¹ to about 10⁵M⁻¹s⁻¹;or from about 10⁵M⁻¹s⁻¹ to about 10⁶M⁻¹s⁻¹, as measured by surfaceplasmon resonance.

In another embodiment, the binding protein has an off rate constant(K_(off)) for one or more targets of at most about 10⁻³s⁻¹; at mostabout 10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹; or at most about 10⁻⁶s⁻¹, asmeasured by surface plasmon resonance. In an embodiment, the bindingprotein has an off rate constant (K_(off)) to one or more targets ofabout 10⁻³s⁻¹ to about 10⁻⁴s⁻¹; of about 10⁻⁴s⁻¹ to about 10⁻⁵s⁻¹; or ofabout 10⁻⁵s⁻¹ to about 10⁻⁶e, as measured by surface plasmon resonance.

In another embodiment, the binding protein has a dissociation constant(K_(d)) to one or more targets of at most about 10⁻⁷M; at most about10⁻⁸M; at most about 10⁻⁹M; at most about 10⁻¹⁰M; at most about 10⁻¹¹M;at most about 10⁻¹²M; or at most 10⁻¹³M. In an embodiment, the bindingprotein has a dissociation constant (K_(d)) to its targets of about10⁻⁷M to about 10⁻⁸M; of about 10⁻⁸M to about 10⁻⁹M; of about 10⁻⁹M toabout 10⁻¹⁹M; of about 10⁻¹⁹M to about 10⁻¹¹M; of about 10⁻¹¹M to about10⁻¹²M; or of about 10¹² to M about 10⁻¹³M.

In another embodiment, the binding protein is a conjugate furthercomprising an agent. In an embodiment, the agent is an immunoadhesionmolecule, an imaging agent, a therapeutic agent, or a cytotoxic agent.In an embodiment, the imaging agent is a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, or biotin. In another embodiment, the radiolabel is ³H,¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm. In yetanother embodiment, the therapeutic or cytotoxic agent is ananti-metabolite, an alkylating agent, an antibiotic, a growth factor, acytokine, an anti-angiogenic agent, an anti-mitotic agent, ananthracycline, toxin, or an apoptotic agent.

In another embodiment, the binding protein is a crystallized bindingprotein and exists as a crystal. In an embodiment, the crystal is acarrier-free pharmaceutical controlled release crystal. In anotherembodiment, the crystallized binding protein has a greater half life invivo than the soluble counterpart of the binding protein. In yet anotherembodiment, the crystallized binding protein retains biologicalactivity.

In another embodiment, the binding protein described herein isglycosylated. For example, the glycosylation pattern is a humanglycosylation pattern.

An isolated nucleic acid encoding any one of the binding proteinsdisclosed herein is also provided. A further embodiment provides avector comprising the isolated nucleic acid disclosed herein wherein thevector is pcDNA; pTT (Durocher et al. (2002) Nucleic Acids Res. 30(2);pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima andNagata (1990) Nucleic Acids Res. 18(17); pBV; pJV; pcDNA3.1 TOPO; pEF6TOPO; pBOS; pHybE; or pBJ. In an embodiment, the vector is a vectordisclosed in US Patent Publication No. 20090239259.

In another aspect, a host cell is transformed with the vector disclosedherein. In an embodiment, the host cell is a prokaryotic cell, forexample, E. coli. In another embodiment, the host cell is a eukaryoticcell, for example, a protist cell, an animal cell, a plant cell, or afungal cell. In an embodiment, the host cell is a mammalian cellincluding, but not limited to, CHO, COS, NS0, SP2, PER.C6, or a fungalcell, such as Saccharomyces cerevisiae, or an insect cell, such as Sf9.In an embodiment, two or more binding proteins, e.g., with differentspecificities, are produced in a single recombinant host cell. Forexample, the expression of a mixture of antibodies has been calledOligoclonics™ (Merus B. V., The Netherlands) U.S. Pat. Nos. 7,262,028and 7,429,486.

A method of producing a binding protein disclosed herein comprisingculturing any one of the host cells disclosed herein in a culture mediumunder conditions sufficient to produce the binding protein is provided.In an embodiment, 50%-75% of the binding protein produced by this methodis a dual specific tetravalent binding protein. In another embodiment,75%-90% of the binding protein produced by this method is a dualspecific tetravalent binding protein. In another embodiment, 90%-95% ofthe binding protein produced is a dual specific tetravalent bindingprotein.

One embodiment provides a composition for the release of a bindingprotein wherein the composition comprises a crystallized bindingprotein, an ingredient, and at least one polymeric carrier. In anembodiment, the polymeric carrier is poly (acrylic acid), a poly(cyanoacrylate), a poly (amino acid), a poly (anhydride), a poly(depsipeptide), a poly (ester), poly (lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly(caprolactone), poly (dioxanone), poly (ethylene glycol), poly((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], a poly(ortho ester), poly (vinyl alcohol), poly (vinylpyrrolidone), a maleicanhydride-alkyl vinyl ether copolymer, a pluronic polyol, albumin,alginate, cellulose, a cellulose derivative, collagen, fibrin, gelatin,hyaluronic acid, an oligosaccharide, a glycaminoglycan, a sulfatedpolysaccharide, or blends and copolymers thereof. In an embodiment, theingredient is albumin, sucrose, trehalose, lactitol, gelatin,hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol, orpolyethylene glycol.

Another embodiment provides a method for treating a mammal comprisingthe step of administering to the mammal an effective amount of acomposition disclosed herein.

A pharmaceutical composition comprising a binding protein disclosedherein and a pharmaceutically acceptable carrier is provided. In afurther embodiment, the pharmaceutical composition comprises at leastone additional therapeutic agent for treating a disorder. For example,the additional agent may be a therapeutic agent, an imaging agent, acytotoxic agent, an angiogenesis inhibitor (including but not limited toan anti-VEGF antibody or a VEGF-trap), a kinase inhibitor (including butnot limited to a KDR and a TIE-2 inhibitor), a co-stimulation moleculeblocker (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig,anti-CD20), an adhesion molecule blocker (including but not limited toan anti-LFA-1 antibody, an anti-E/L selectin antibody, a small moleculeinhibitor), an anti-cytokine antibody or functional fragment thereof(including but not limited to an anti-IL-18, an anti-TNF, and ananti-IL-6/cytokine receptor antibody), methotrexate, cyclosporin,rapamycin, FK506, a detectable label or reporter, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog, a cytokine,or a cytokine antagonist.

A method for treating a human subject suffering from a disorder in whichthe target, or targets, capable of being bound by the binding proteindisclosed herein is detrimental, comprising administering to the humansubject a binding protein disclosed herein such that the activity of thetarget, or targets, in the human subject is inhibited and one or moresymptoms is alleviated or treatment is achieved is provided. The bindingproteins provided herein can be used to treat humans suffering fromautoimmune diseases such as, for example, those associated withinflammation. In an embodiment, the binding proteins provided herein orantigen-binding portions thereof, are used to treat asthma, allergies,allergic lung disease, allergic rhinitis, atopic dermatitis, chronicobstructive pulmonary disease (COPD), fibrosis, cystic fibrosis (CF),fibrotic lung disease, idiopathic pulmonary fibrosis, liver fibrosis,lupus, hepatitis B-related liver diseases and fibrosis, sepsis, systemiclupus erythematosus (SLE), glomerulonephritis, inflammatory skindiseases, psoriasis, diabetes, insulin dependent diabetes mellitus,infectious diseases caused by HIV, inflammatory bowel disease (IBD),ulcerative colitis (UC), Crohn's disease (CD), rheumatoid arthritis(RA), osteoarthritis (OA), multiple sclerosis (MS), graft-versus-hostdisease (GVHD), transplant rejection, ischemic heart disease (IHD),celiac disease, contact hypersensitivity, alcoholic liver disease,Behcet's disease, atherosclerotic vascular disease, occular surfaceinflammatory diseases, or Lyme disease.

In another embodiment, the disorder or condition to be treated comprisesthe symptoms caused by viral infection in a human which is caused by,for example, HIV, the human rhinovirus, an enterovirus, a coronavirus, aherpes virus, an influenza virus, a parainfluenza virus, a respiratorysyncytial virus or an adenovirus.

The binding proteins provided herein can be used to treat neurologicaldisorders. In an embodiment, the binding proteins provided herein, orantigen-binding portions thereof, are used to treat neurodegenerativediseases and conditions involving neuronal regeneration and spinal cordinjury.

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods disclosed herein include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

Another embodiment provides for the use of the binding protein in thetreatment of a disease or disorder, wherein said disease or disorder isrheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, septicarthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,ulcerative colitis, inflammatory bowel disease, insulin dependentdiabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis,dermatitis scleroderma, graft versus host disease, organ transplantrejection, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, infectious diseases, parasitic diseases,acquired immunodeficiency syndrome, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, Addison's disease, sporadic, polyglandular deficiency type Iand polyglandular deficiency type II, Schmidt's syndrome, adult (acute)respiratory distress syndrome, alopecia, alopecia greata, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, atheromatous disease/arteriosclerosis, atopic allergy,autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus,pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombspositive haemolytic anaemia, acquired pernicious anaemia, juvenilepernicious anaemia, myalgic encephalitis/Royal Free Disease, chronicmucocutaneous candidiasis, giant cell arteritis, primary sclerosinghepatitis, cryptogenic autoimmune hepatitis, acquired immunodeficiencyrelated diseases, hepatitis B, hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, female infertility, ovarian failure, premature ovarianfailure, fibrotic lung disease, cryptogenic fibrosing alveolitis,post-inflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease, Sjögren'sdisease associated lung disease, ankylosing spondylitis associated lungdisease, vasculitic diffuse lung disease, haemosiderosis associated lungdisease, drug-induced interstitial lung disease, fibrosis, radiationfibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia,lymphocytic infiltrative lung disease, postinfectious interstitial lungdisease, gouty arthritis, autoimmune hepatitis, type-1 autoimmunehepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmunehepatitis (anti-LKM antibody hepatitis), autoimmune mediatedhypoglycaemia, type B insulin resistance with acanthosis nigricans,hypoparathyroidism, acute immune disease associated with organtransplantation, chronic immune disease associated with organtransplantation, osteoarthrosis, primary sclerosing cholangitis,psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmuneneutropaenia, renal disease NOS, glomerulonephritides, microscopicvasulitis of the kidneys, lyme disease, discoid lupus erythematosus,male infertility idiopathic or NOS, sperm autoimmunity, multiplesclerosis (all subtypes), sympathetic ophthalmia, pulmonary hypertensionsecondary to connective tissue disease, Goodpasture's syndrome,pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever,rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren'ssyndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia,idiopathic thrombocytopaenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis, vitiligo acute liver disease,chronic liver diseases, alcoholic cirrhosis, alcohol-induced liverinjury, choleosatatis, idiosyncratic liver disease, drug-inducedhepatitis, non-alcoholic steatohepatitis, allergy and asthma, group Bstreptococci (GBS) infection, mental disorders, depression,schizophrenia, Th2 Type and TM Type mediated diseases, acute and chronicpain, different forms of pain, cancers, lung cancer, breast cancer,stomach cancer, bladder cancer, colon cancer, pancreatic cancer, ovariancancer, prostate cancer, rectal cancer, hematopoietic malignancies,leukemia, lymphoma, Abetalipoprotemia, acrocyanosis, acute and chronicparasitic or infectious processes, acute leukemia, acute lymphoblasticleukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterialinfection, acute pancreatitis, acute renal failure, adenocarcinomas,aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis,allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis,allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic lateralsclerosis, anemia, angina pectoris, anterior horn cell degeneration,anti cd3 therapy, antiphospholipid syndrome, anti-receptorhypersensitivity reactions, aortic and peripheral aneuryisms, aorticdissection, arterial hypertension, arteriosclerosis, arteriovenousfistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrialflutter, atrioventricular block, B cell lymphoma, bone graft rejection,bone marrow transplant (BMT) rejection, bundle branch block, Burkitt'slymphoma, burns, cardiac arrhythmias, cardiac stun syndrome, cardiactumors, cardiomyopathy, cardiopulmonary bypass inflammation response,cartilage transplant rejection, cerebellar cortical degenerations,cerebellar disorders, chaotic or multifocal atrial tachycardia,chemotherapy associated disorders, chronic myelocytic leukemia (CML),chronic alcoholism, chronic inflammatory pathologies, chroniclymphocytic leukemia (CLL), chronic obstructive pulmonary disease(COPD), chronic salicylate intoxication, colorectal carcinoma,congestive heart failure, conjunctivitis, contact dermatitis, corpulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culturenegative sepsis, cystic fibrosis, cytokine therapy associated disorders,dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever,dermatitis, dermatologic conditions, diabetes, diabetes mellitus,diabetic ateriosclerotic disease, diffuse Lewy body disease, dilatedcongestive cardiomyopathy, disorders of the basal ganglia, Down'ssyndrome in middle age, drug-induced movement disorders induced by drugswhich block CNS dopamine receptors, drug sensitivity, eczema,encephalomyelitis, endocarditis, endocrinopathy, epiglottitis,epstein-barr virus infection, erythromelalgia, extrapyramidal andcerebellar disorders, familial hematophagocytic lymphohistiocytosis,fetal thymus implant rejection, Friedreich's ataxia, functionalperipheral arterial disorders, fungal sepsis, gas gangrene, gastriculcer, glomerular nephritis, graft rejection of any organ or tissue,gram negative sepsis, gram positive sepsis, granulomas due tointracellular organisms, hairy cell leukemia, Hallervorden-Spatzdisease, Hashimoto's thyroiditis, hay fever, heart transplant rejection,hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolyticthrombocytopenic purpura, hemorrhage, hepatitis A, His bundlearrythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignant lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti.system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (MencelDejerine-Thomas Shi-Drager and Machado-Joseph), mycobacterium aviumintracellulare, mycobacterium tuberculosis, myelodyplastic syndrome,myocardial infarction, myocardial ischemic disorders, nasopharyngealcarcinoma, neonatal chronic lung disease, nephritis, nephrosis,neurodegenerative diseases, neurogenic muscular atrophies, neutropenicfever, non-hodgkins lymphoma, occlusion of the abdominal aorta and itsbranches, occulsive arterial disorders, okt3 therapy,orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherlosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, progressive supranucleopalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, seniledementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, subacute sclerosingpanencephalitis, syncope, syphilis of the cardiovascular system,systemic anaphalaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, valvular heartdiseases, varicose veins, vasculitis, venous diseases, venousthrombosis, ventricular fibrillation, viral and fungal infections, vitalencephalitis/aseptic meningitis, vital-associated hemaphagocyticsyndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograftrejection of any organ or tissue, acute coronary syndromes, acuteidiopathic polyneuritis, acute inflammatory demyelinatingpolyradiculoneuropathy, acute ischemia, adult Still's disease,anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia,atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmunedisorder associated with streptococcus infection, autoimmuneenteropathy, autoimmune hearing loss, autoimmune lymphoproliferativesyndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarianfailure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovasculardisease, catastrophic antiphospholipid syndrome, celiac disease,cervical spondylosis, chronic ischemia, cicatricial pemphigoid,clinically isolated syndrome (cis) with risk for multiple sclerosis,childhood onset psychiatric disorder, dacryocystitis, dermatomyositis,diabetic retinopathy, disk herniation, disk prolaps, drug induced immunehemolytic anemia, endometriosis, endophthalmitis, episcleritis, erythemamultiforme, erythema multiforme major, gestational pemphigoid,Guillain-Barré syndrome (GBS), Hughes syndrome, idiopathic Parkinson'sdisease, idiopathic interstitial pneumonia, IgE-mediated allergy, immunehemolytic anemia, inclusion body myositis, infectious ocularinflammatory disease, inflammatory demyelinating disease, inflammatoryheart disease, inflammatory kidney disease, IPF/UIP, iritis, keratitis,keratojuntivitis sicca, Kussmaul disease or Kussmaul-Meier disease,Landry's paralysis, Langerhan's cell histiocytosis, livedo reticularis,macular degeneration, microscopic polyangiitis, morbus bechterev, motorneuron disorders, mucous membrane pemphigoid, multiple organ failure,myasthenia gravis, myelodysplastic syndrome, myocarditis, nerve rootdisorders, neuropathy, non-A non-B hepatitis, optic neuritis,osteolysis, pauciarticular JRA, peripheral artery occlusive disease(PAOD), peripheral vascular disease (PVD), peripheral artery, disease(PAD), phlebitis, polyarteritis nodosa (or periarteritis nodosa),polychondritis, poliosis, polyarticular JRA, polyendocrine deficiencysyndrome, polymyositis, polymyalgia rheumatica (PMR), primaryParkinsonism, prostatitis, pure red cell aplasia, primary adrenalinsufficiency, recurrent neuromyelitis optica, restenosis, rheumaticheart disease, sapho (synovitis, acne, pustulosis, hyperostosis, andosteitis), secondary amyloidosis, shock lung, scleritis, sciatica,secondary adrenal insufficiency, silicone associated connective tissuedisease, sneddon-wilkinson dermatosis, spondilitis ankylosans,Stevens-Johnson syndrome (SJS), temporal arteritis, toxoplasmicretinitis, toxic epidermal necrolysis, transverse myelitis, TRAPS (tumornecrosis factor receptor, type 1 allergic reaction, type II diabetes,urticaria, usual interstitial pneumonia (UIP), vasculitis, vernalconjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKHsyndrome), wet macular degeneration, or wound healing.

In an embodiment, the binding proteins, or antigen-binding portionsthereof, are used to treat cancer or in the prevention or inhibition ofmetastases from the tumors described herein either when used alone or incombination with radiotherapy and/or chemotherapeutic agents.

In another aspect, methods of treating a patient suffering from adisorder comprising the step of administering any one of the bindingproteins disclosed herein before, concurrently, or after theadministration of a second agent, are provided. In an embodiment, thesecond agent is budenoside, epidermal growth factor, a corticosteroid,cyclosporin, sulfasalazine, an aminosalicylate, 6-mercaptopurine,azathioprine, metronidazole, a lipoxygenase inhibitor, mesalamine,olsalazine, balsalazide, an antioxidant, a thromboxane inhibitor, anIL-1 receptor antagonist, an anti-IL-1β mAbs, an anti-IL-6 or IL-6receptor mAb, a growth factor, an elastase inhibitor, apyridinyl-imidazole compound, an antibody or agonist of TNF, LT, IL-1,IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23,EMAP-II, GM-CSF, FGF, or PDGF, an antibody to CD2, CD3, CD4, CD8, CD-19,CD25, CD28, CD30, CD40, CD45, CD69, CD90 or a ligand thereof,methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, an NSAID, ibuprofen, prednisolone, a phosphodiesteraseinhibitor, an adenosine agonist, an antithrombotic agent, a complementinhibitor, an adrenergic agent, IRAK, NIK, IKK, p38, a MAP kinaseinhibitor, an IL-1β converting enzyme inhibitor, a TNFα-convertingenzyme inhibitor, a T-cell signalling inhibitor, a metalloproteinaseinhibitor, sulfasalazine, azathioprine, a 6-mercaptopurine, anangiotensin converting enzyme inhibitor, a soluble cytokine receptor, asoluble p55 TNF receptor, a soluble p75 TNF receptor, sIL-1RI, sIL-1RII,sIL-6R, an antiinflammatory cytokine, IL-4, IL-10, IL-11, IL-13, orTG93. In a particular embodiment, the pharmaceutical compositionsdisclosed herein are administered to a patient by parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermaladministration.

Anti-idiotype antibodies to the binding proteins disclosed herein arealso provided. An anti-idiotype antibody includes any protein orpeptide-containing molecule that comprises at least a portion of animmunoglobulin molecule such as, but not limited to, at least onecomplementarily determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into a bindingprotein provided herein.

A method of determining the presence, amount or concentration of IL-13and/or IL-17, or fragment thereof, in a test sample is provided. Themethod comprises assaying the test sample for the antigen, or fragmentthereof, by an immunoassay. The immunoassay (i) employs at least onebinding protein and at least one detectable label and (ii) comprisescomparing a signal generated by the detectable label as a direct orindirect indication of the presence, amount or concentration of theantigen, or fragment thereof, in the test sample to a signal generatedas a direct or indirect indication of the presence, amount orconcentration of the antigen, or fragment thereof, in a control or acalibrator. The calibrator is optionally part of a series of calibratorsin which each of the calibrators differs from the other calibrators inthe series by the concentration of the antigen, or fragment thereof. Themethod can comprise (i) contacting the test sample with at least onecapture agent, which binds to an epitope on the antigen, or fragmentthereof, so as to form a capture agent/antigen, or fragment thereof,complex, (ii) contacting the capture agent/antigen, or fragment thereof,complex with at least one detection agent, which comprises a detectablelabel and binds to an epitope on the antigen, or fragment thereof, thatis not bound by the capture agent, to form a capture agent/antigen, orfragment thereof/detection agent complex, and (iii) determining thepresence, amount or concentration of the antigen, or fragment thereof,in the test sample based on the signal generated by the detectable labelin the capture agent/antigen, or fragment thereof/detection agentcomplex formed in (ii), wherein at least one capture agent and/or atleast one detection agent is the at least one binding protein.

Alternatively, the method can comprise (i) contacting the test samplewith at least one capture agent, which binds to an epitope on theantigen, or fragment thereof, so as to form a capture agent/antigen, orfragment thereof, complex, and simultaneously or sequentially, in eitherorder, contacting the test sample with detectably labeled antigen, orfragment thereof, which can compete with any antigen, or fragmentthereof, in the test sample for binding to the at least one captureagent, wherein any antigen, or fragment thereof, present in the testsample and the detectably labeled antigen compete with each other toform a capture agent/antigen, or fragment thereof, complex and a captureagent/detectably labeled antigen, or fragment thereof, complex,respectively, and (ii) determining the presence, amount or concentrationof the antigen, or fragment thereof, in the test sample based on thesignal generated by the detectable label in the capture agent/detectablylabeled antigen, or fragment thereof, complex formed in (ii), wherein atleast one capture agent is the at least one binding protein and whereinthe signal generated by the detectable label in the captureagent/detectably labeled antigen, or fragment thereof, complex isinversely proportional to the amount or concentration of antigen, orfragment thereof, in the test sample.

The test sample can be from a patient, in which case the method canfurther comprise diagnosing, prognosticating, or assessing the efficacyof therapeutic/prophylactic treatment of the patient. If the methodfurther comprises assessing the efficacy of therapeutic/prophylactictreatment of the patient, the method optionally further comprisesmodifying the therapeutic/prophylactic treatment of the patient asneeded to improve efficacy. The method can be adapted for use in anautomated system or a semi-automated system. Accordingly, the methodsdescribed herein also can be used to determine whether or not a subjecthas or is at risk of developing a given disease, disorder or condition.Specifically, such a method can comprise the steps of:

(a) determining the concentration or amount in a test sample from asubject of analyte, or fragment thereof, (e.g., using the methodsdescribed herein, or methods known in the art); and

(b) comparing the concentration or amount of analyte, or fragmentthereof, determined in step (a) with a predetermined level, wherein, ifthe concentration or amount of analyte determined in step (a) isfavorable with respect to a predetermined level, then the subject isdetermined not to have or be at risk for a given disease, disorder orcondition. However, if the concentration or amount of analyte determinedin step (a) is unfavorable with respect to the predetermined level, thenthe subject is determined to have or be at risk for a given disease,disorder or condition.

Additionally, provided herein is method of monitoring the progression ofdisease in a subject. Optimally the method comprising the steps of:

(a) determining the concentration or amount in a test sample from asubject of analyte;

(b) determining the concentration or amount in a later test sample fromthe subject of analyte; and

(c) comparing the concentration or amount of analyte as determined instep (b) with the concentration or amount of analyte determined in step(a), wherein if the concentration or amount determined in step (b) isunchanged or is unfavorable when compared to the concentration or amountof analyte determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened. By comparison, ifthe concentration or amount of analyte as determined in step (b) isfavorable when compared to the concentration or amount of analyte asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved.

Optionally, the method further comprises comparing the concentration oramount of analyte as determined in step (b), for example, with apredetermined level. Further, optionally the method comprises treatingthe subject with one or more pharmaceutical compositions for a period oftime if the comparison shows that the concentration or amount of analyteas determined in step (b), for example, is unfavorably altered withrespect to the predetermined level.

Also provided is a kit for assaying a test sample for IL-13 and/orIL-17, or fragment thereof. The kit comprises at least one component forassaying the test sample for an antigen, or fragment thereof, andinstructions for assaying the test sample for an antigen, or fragmentthereof, wherein the at least one component includes at least onecomposition comprising the binding protein disclosed herein, wherein thebinding protein is optionally detectably labeled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of Dual Variable Domain (DVD)binding protein constructs.

DETAILED DESCRIPTION

Multivalent and/or multispecific binding proteins capable of bindingIL13 and/or IL-17 are provided. Dual variable domain binding proteins(DVD binding proteins) or dual variable domain immunoglobulins(DVD-Ig™), and pharmaceutical compositions thereof, as well as nucleicacids, recombinant expression vectors and host cells for making such DVDbinding proteins are also provided. Methods of using the DVD bindingproteins to detect specific antigens, either in vitro or in vivo arealso provided.

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting.

Generally, nomenclatures used in connection with cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well known and commonly used in the art. The methods andtechniques provided herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

That the disclosure may be more readily understood, select terms aredefined below.

The term “antibody” refers to an immunoglobulin (Ig) molecule, which isgenerally comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains, or a functional fragment, mutant, variant, orderivative thereof, that retains the epitope binding features of an Igmolecule. Such fragment, mutant, variant, or derivative antibody formatsare known in the art. In an embodiment of a full-length antibody, eachheavy chain is comprised of a heavy chain variable region (VH) and aheavy chain constant region (CH). The CH is comprised of three domains,CH1, CH2 and CH3. Each light chain is comprised of a light chainvariable region (VL) and a light chain constant region (CL). The CL iscomprised of a single CL domain. The VH and VL can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs). Generally, each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2), or subclass.

The term “bispecific antibody” refers to an antibody that binds oneantigen (or epitope) on one of its two binding arms (one pair of HC/LC),and binds a different antigen (or epitope) on its second binding arm (adifferent pair of HC/LC). A bispecific antibody has two distinct antigenbinding arms (in both specificity and CDR sequences), and is monovalentfor each antigen to which it binds. Bispecific antibodies include thosegenerated by quadroma technology (Milstein and Cuello (1983) Nature305(5934): 537-40), by chemical conjugation of two different monoclonalantibodies (Staerz et al. (1985) Nature 314(6012): 628-31), or byknob-into-hole or similar approaches which introduces mutations in theFc region (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90(14):6444-6448).

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Exemplary affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen. Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. (1992) BioTechnology 10:779-783 describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by Barbas et al. (1994) Proc.Nat. Acad. Sci. USA 91:3809-3813; Schier et al. (1995) Gene 169:147-155;Yelton et al. (1995) J. Immunol. 155:1994-2004; Jackson et al. (1995) J.Immunol. 154(7):3310-9; Hawkins et al. (1992) J. Mol. Biol. 226:889-896and mutation at selective mutagenesis positions, contact orhypermutation positions with an activity enhancing amino acid residue asdescribed in U.S. Pat. No. 6,914,128.

The term “CDR-grafted antibody” refers to an antibody that comprisesheavy and light chain variable region sequences in which the sequencesof one or more of the CDR regions of VH and/or VL are replaced with CDRsequences of another antibody. For example, the two antibodies can befrom different species, such as antibodies having murine heavy and lightchain variable regions in which one or more of the murine CDRs has beenreplaced with human CDR sequences.

The term “humanized antibody” refers to an antibody from a non-humanspecies that has been altered to be more “human-like”, i.e., moresimilar to human germline sequences. One type of humanized antibody is aCDR-grafted antibody, in which non-human CDR sequences are introducedinto human VH and VL sequences to replace the corresponding human CDRsequences. A “humanized antibody” is also an antibody or a variant,derivative, analog or fragment thereof that comprises framework region(FR) sequences having substantially (e.g., at least 80%, at least 85%,at least 90%, at least 95%, at least 98% or at least 99% identity to)the amino acid sequence of a human antibody and at least one CDR havingsubstantially the amino acid sequence of a non-human antibody. Ahumanized antibody may comprise substantially all of at least one, andtypically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in whichthe sequence of all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin (i.e., donor antibody) and thesequence of all or substantially all of the FR regions are those of ahuman immunoglobulin. The humanized antibody also may include the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain. In an embodiment, ahumanized antibody also comprises at least a portion of a humanimmunoglobulin Fc region. In some embodiments, a humanized antibody onlycontains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In some embodiments, ahumanized antibody only contains a humanized variable domain of a lightchain and/or humanized variable domain of a heavy chain. In someembodiments, a humanized antibody contains a light chain as well as atleast the variable domain of a heavy chain. In some embodiments, ahumanized antibody contains a heavy chain as well as at least thevariable domain of a light chain.

The terms “dual variable domain binding protein” and “dual variabledomain immunoglobulin” refer to a binding protein that has two variabledomains in each of its two binding arms (e.g., a pair of HC/LC) (see PCTPublication No. WO 02/02773), each of which is able to bind to anantigen. In an embodiment, each variable domain binds different antigensor epitopes. In another embodiment, each variable domain binds the sameantigen or epitope. In another embodiment, a dual variable domainbinding protein has two identical antigen binding arms, with identicalspecificity and identical CDR sequences, and is bivalent for eachantigen to which it binds. In an embodiment, the DVD binding proteinsmay be monospecific, i.e., capable of binding one antigen ormultispecific, i.e., capable of binding two or more antigens. DVDbinding proteins comprising two heavy chain DVD polypeptides and twolight chain DVD polypeptides are referred to as a DVD-Ig™. In anembodiment, each half of a four chain DVD binding protein comprises aheavy chain DVD polypeptide, and a light chain DVD polypeptide, and twoantigen binding sites. In an embodiment, each binding site comprises aheavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site.

The term “antiidiotypic antibody” refers to an antibody raised againstthe amino acid sequence of the antigen combining site of anotherantibody. Antiidiotypic antibodies may be administered to enhance animmune response against an antigen.

The term “biological activity” refers to any one or more biologicalproperties of a molecule (whether present naturally as found in vivo, orprovided or enabled by recombinant means). Biological propertiesinclude, but are not limited to, binding a receptor, inducing cellproliferation, inhibiting cell growth, inducing other cytokines,inducing apoptosis, and enzymatic activity.

The term “neutralizing” refers to counteracting the biological activityof an antigen when a binding protein specifically binds to the antigen.In an embodiment, the neutralizing binding protein binds to an antigen(e.g., a cytokine) and reduces its biologically activity by at leastabout 20%, 40%, 60%, 80%, 85% or more.

“Specificity” refers to the ability of a binding protein to selectivelybind an antigen.

“Affinity” is the strength of the interaction between a binding proteinand an antigen, and is determined by the sequence of the CDRs of thebinding protein as well as by the nature of the antigen, such as itssize, shape, and/or charge. Binding proteins may be selected foraffinities that provide desired therapeutic end-points while minimizingnegative side-effects. Affinity may be measured using methods known toone skilled in the art (US 20090311253).

The term “potency” refers to the ability of a binding protein to achievea desired effect, and is a measurement of its therapeutic efficacy.Potency may be assessed using methods known to one skilled in the art(US 20090311253).

The term “cross-reactivity” refers to the ability of a binding proteinto bind a target other than that against which it was raised. Generally,a binding protein will bind its target tissue(s)/antigen(s) with anappropriately high affinity, but will display an appropriately lowaffinity for non-target normal tissues. Individual binding proteins aregenerally selected to meet two criteria. (1) Tissue staining appropriatefor the known expression of the antibody target. (2) Similar stainingpattern between human and tox species (mouse and cynomolgus monkey)tissues from the same organ. These and other methods of assessingcross-reactivity are known to one skilled in the art (US 20090311253).

The term “biological function” refers the specific in vitro or in vivoactions of a binding protein. Binding proteins may target severalclasses of antigens and achieve desired therapeutic outcomes throughmultiple mechanisms of action. Binding proteins may target solubleproteins, cell surface antigens, as well as extracellular proteindeposits. Binding proteins may agonize, antagonize, or neutralize theactivity of their targets. Binding proteins may assist in the clearanceof the targets to which they bind, or may result in cytotoxicity whenbound to cells. Portions of two or more antibodies may be incorporatedinto a multivalent format to achieve distinct functions in a singlebinding protein molecule. The in vitro assays and in vivo models used toassess biological function are known to one skilled in the art (US20090311253).

A “stable” binding protein is one in which the binding proteinessentially retains its physical stability, chemical stability and/orbiological activity upon storage. A multivalent binding protein that isstable in vitro at various temperatures for an extended period of timeis desirable. Methods of stabilizing binding proteins and assessingtheir stability at various temperatures are known to one skilled in theart (US 20090311253).

The term “solubility” refers to the ability of a protein to remaindispersed within an aqueous solution. The solubility of a protein in anaqueous formulation depends upon the proper distribution of hydrophobicand hydrophilic amino acid residues, and therefore, solubility cancorrelate with the production of correctly folded proteins. A personskilled in the art will be able to detect an increase or decrease insolubility of a binding protein using routine HPLC techniques andmethods known to one skilled in the art (US 20090311253).

Binding proteins may be produced using a variety of host cells or may beproduced in vitro, and the relative yield per effort determines the“production efficiency.” Factors influencing production efficiencyinclude, but are not limited to, host cell type (prokaryotic oreukaryotic), choice of expression vector, choice of nucleotide sequence,and methods employed. The materials and methods used in binding proteinproduction, as well as the measurement of production efficiency, areknown to one skilled in the art (US 20090311253).

The term “immunogenicity” means the ability of a substance to induce animmune response. Administration of a therapeutic binding protein mayresult in a certain incidence of an immune response. Potential elementsthat might induce immunogenicity in a multivalent format may be analyzedduring selection of the parental antibodies, and steps to reduce suchrisk can be taken to optimize the parental antibodies prior toincorporating their sequences into a multivalent binding protein format.Methods of reducing the immunogenicity of antibodies and bindingproteins are known to one skilled in the art (US 20090311253).

The terms “label” and “detectable label” mean a moiety attached to amember of a specific binding pair, such as an antibody or its analyte torender a reaction (e.g., binding) between the members of the specificbinding pair, detectable. The labeled member of the specific bindingpair is referred to as “detectably labeled.” Thus, the term “labeledbinding protein” refers to a protein with a label incorporated thatprovides for the identification of the binding protein. In anembodiment, the label is a detectable marker that can produce a signalthat is detectable by visual or instrumental means, e.g., incorporationof a radiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (e.g., streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). Examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm); chromogens, fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags); and magnetic agents, such as gadoliniumchelates. Representative examples of labels commonly employed forimmunoassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein. Inthis regard, the moiety itself may not be detectably labeled but maybecome detectable upon reaction with yet another moiety.

The term “conjugate” refers to a binding protein, such as an antibody,that is chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” includes a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials. In an embodiment, thetherapeutic or cytotoxic agents include, but are not limited to,pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. When employed in the contextof an immunoassay, the conjugate antibody may be a detectably labeledantibody used as the detection antibody.

The terms “crystal” and “crystallized” refer to a binding protein (e.g.,an antibody), or antigen binding portion thereof, that exists in theform of a crystal. Crystals are one form of the solid state of matter,which is distinct from other forms such as the amorphous solid state orthe liquid crystalline state. Crystals are composed of regular,repeating, three-dimensional arrays of atoms, ions, molecules (e.g.,proteins such as antibodies), or molecular assemblies (e.g.,antigen/antibody complexes). These three-dimensional arrays are arrangedaccording to specific mathematical relationships that arewell-understood in the field. The fundamental unit, or building block,that is repeated in a crystal is called the asymmetric unit. Repetitionof the asymmetric unit in an arrangement that conforms to a given,well-defined crystallographic symmetry provides the “unit cell” of thecrystal. Repetition of the unit cell by regular translations in allthree dimensions provides the crystal. See Giege, R. and Ducruix, A.Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS AND PROTEINS, A PRACTICALAPPROACH, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y.,(1999).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Other vectors include RNA vectors. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”(or simply, “expression vectors”). In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, other forms of expression vectors are also included, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions. A groupof pHybE vectors (U.S. Patent Application Ser. No. 61/021,282) were usedfor parental antibody and DVD-binding protein cloning. V1, derived frompJP183; pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVDheavy chains with a wildtype constant region. V2, derived from pJP191;pHybE-hCk V3, was used for cloning of antibody and DVD light chains witha kappa constant region. V3, derived from pJP192; pHybE-hCl V2, was usedfor cloning of antibody and DVDs light chains with a lambda constantregion. V4, built with a lambda signal peptide and a kappa constantregion, was used for cloning of DVD light chains with a lambda-kappahybrid V domain. V5, built with a kappa signal peptide and a lambdaconstant region, was used for cloning of DVD light chains with akappa-lambda hybrid V domain. V7, derived from pJP183;pHybE-hCg1,z,non-a V2, was used for cloning of antibody and DVD heavychains with a (234,235 AA) mutant constant region.

The terms “recombinant host cell” or “host cell” refer to a cell intowhich exogenous DNA has been introduced. Such terms refer not only tothe particular subject cell, but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. In an embodiment, host cellsinclude prokaryotic and eukaryotic cells. In an embodiment, eukaryoticcells include protist, fungal, plant and animal cells. In anotherembodiment, host cells include but are not limited to the prokaryoticcell line E. Coli; mammalian cell lines CHO, HEK 293, COS, NS0, SP2 andPER.C6; the insect cell line Sf9; and the fungal cell Saccharomycescerevisiae.

The term “transfection” encompasses a variety of techniques commonlyused for the introduction of exogenous nucleic acid (e.g., DNA) into ahost cell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like.

The term “cytokine” refers to a protein released by one cell populationthat acts on another cell population as an intercellular mediator. Theterm “cytokine” includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

The term “biological sample” means a quantity of a substance from aliving thing or formerly living thing. Such substances include, but arenot limited to, blood, (e.g., whole blood), plasma, serum, urine,amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

The term “component” refers to an element of a composition. In relationto a diagnostic kit, for example, a component may be a capture antibody,a detection or conjugate antibody, a control, a calibrator, a series ofcalibrators, a sensitivity panel, a container, a buffer, a diluent, asalt, an enzyme, a co-factor for an enzyme, a detection reagent, apretreatment reagent/solution, a substrate (e.g., as a solution), a stopsolution, and the like that can be included in a kit for assay of a testsample. Thus, a “component” can include a polypeptide or other analyteas above, that is immobilized on a solid support, such as by binding toan anti-analyte (e.g., anti-polypeptide) antibody. Some components canbe in solution or lyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not analyte (“negativecontrol”) or to contain analyte (“positive control”). A positive controlcan comprise a known concentration of analyte. “Control,” “positivecontrol,” and “calibrator” may be used interchangeably herein to referto a composition comprising a known concentration of analyte. A“positive control” can be used to establish assay performancecharacteristics and is a useful indicator of the integrity of reagents(e.g., analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(e.g., antibodies employed, etc.). It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) may be generally applicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte (e.g.,polypeptide of interest) may entail release of the analyte from anyendogenous binding proteins present in the sample. A pretreatmentreagent may be homogeneous (not requiring a separation step) orheterogeneous (requiring a separation step). With use of a heterogeneouspretreatment reagent there is removal of any precipitated analytebinding proteins from the test sample prior to proceeding to the nextstep of the assay.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

The term “specific binding partner” is a member of a specific bindingpair. A specific binding pair comprises two different molecules thatspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding, otherspecific binding pairs can include biotin and avidin (or streptavidin),carbohydrates and lectins, complementary nucleotide sequences, effectorand receptor molecules, cofactors and enzymes, enzyme inhibitors andenzymes, and the like. Furthermore, specific binding pairs can includemembers that are analogs of the original specific binding members, forexample, an analyte-analog. Immunoreactive specific binding membersinclude antigens, antigen fragments, and antibodies, includingmonoclonal and polyclonal antibodies as well as complexes, fragments,and variants (including fragments of variants) thereof, whether isolatedor recombinantly produced.

The term “Fc region” defines the C-terminal region of an immunoglobulinheavy chain, which may be generated by papain digestion of an intactantibody. The Fc region may be a native sequence Fc region or a variantFc region. The Fc region of an immunoglobulin generally comprises twoconstant domains, a CH2 domain and a CH3 domain, and optionallycomprises a CH4 domain. Replacements of amino acid residues in the Fcportion to alter antibody effector function are known in the art (e.g.,U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc region mediates severalimportant effector functions, e.g., cytokine induction, antibodydependent cell mediated cytotoxicity (ADCC), phagocytosis, complementdependent cytotoxicity (CDC), and half-life/clearance rate of antibodyand antigen-antibody complexes. In some cases these effector functionsare desirable for a therapeutic immunoglobulin but in other cases mightbe unnecessary or even deleterious, depending on the therapeuticobjectives.

The term “antigen-binding portion” of a binding protein means one ormore fragments of a binding protein (e.g., an antibody) that retain theability to specifically bind to an antigen. The antigen-binding portionof a binding protein can be performed by fragments of a full-lengthantibody, as well as bispecific, dual specific, or multi-specificformats; specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an binding protein include (i) an Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) an F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) anFd fragment consisting of the VH and CH1 domains; (iv) an Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment, which comprises a single variable domain; and (vi) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, encoded by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. In addition, single chain antibodies also include “linearantibodies” comprising a pair of tandem Fv segments (VH-CH1-VH-CH1)which, together with complementary light chain polypeptides, form a pairof antigen binding regions.

The term “multivalent binding protein” means a binding proteincomprising two or more antigen binding sites. In an embodiment, themultivalent binding protein is engineered to have three or more antigenbinding sites, and is not a naturally occurring antibody. The term“multispecific binding protein” refers to a binding protein capable ofbinding two or more related or unrelated targets. In an embodiment, thedual variable domain (DVD) binding proteins provided herein comprise twoor more antigen binding sites and are tetravalent or multivalent bindingproteins.

The term “linker” means an amino acid residue or a polypeptidecomprising two or more amino acid residues joined by peptide bonds thatare used to link two polypeptides (e.g., two VH or two VL domains). Suchlinker polypeptides are well known in the art (see, e.g., Holliger etal. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994)Structure 2:1121-1123).

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci.190:382-391 and, Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

The term “CDR” means a complementarity determining region within animmunoglobulin variable region sequence. There are three CDRs in each ofthe variable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the heavy and light chainvariable regions. The term “CDR set” refers to a group of three CDRsthat occur in a single variable region capable of binding the antigen.The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal. (1987) and (1991)) not only provides an unambiguous residuenumbering system applicable to any variable region of an antibody, butalso provides precise residue boundaries defining the three CDRs. TheseCDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothiaand Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature342:877-883) found that certain sub-portions within Kabat CDRs adoptnearly identical peptide backbone conformations, despite having greatdiversity at the level of amino acid sequence. These sub-portions weredesignated as L1, L2 and L3 or H1, H2 and H3 where the “L” and the “H”designates the light chain and the heavy chain regions, respectively.These regions may be referred to as Chothia CDRs, which have boundariesthat overlap with Kabat CDRs. Other boundaries defining CDRs overlappingwith the Kabat CDRs have been described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J. Mol. Biol. 262(5):732-45). Still otherCDR boundary definitions may not strictly follow one of the hereinsystems, but will nonetheless overlap with the Kabat CDRs, although theymay be shortened or lengthened in light of prediction or experimentalfindings that particular residues or groups of residues or even entireCDRs do not significantly impact antigen binding. The methods usedherein may utilize CDRs defined according to any of these systems,although certain embodiments use Kabat or Chothia defined CDRs.

The term “epitope” means a region of an antigen that is bound by abinding protein, e.g., a polypeptide and/or other determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. In an embodiment, an epitope comprises the amino acidresidues of a region of an antigen (or fragment thereof) known to bindto the complementary site on the specific binding partner. An antigenicfragment can contain more than one epitope. In certain embodiments, abinding protein specifically binds an antigen when it recognizes itstarget antigen in a complex mixture of proteins and/or macromolecules.Binding proteins “bind to the same epitope” if the antibodiescross-compete (one prevents the binding or modulating effect of theother). In addition, structural definitions of epitopes (overlapping,similar, identical) are informative; and functional definitionsencompass structural (binding) and functional (modulation, competition)parameters. Different regions of proteins may perform differentfunctions. For example specific regions of a cytokine interact with itscytokine receptor to bring about receptor activation whereas otherregions of the protein may be required for stabilizing the cytokine. Toabrogate the negative effects of cytokine signaling, the cytokine may betargeted with a binding protein that binds specifically to the receptorinteracting region(s), thereby preventing the binding of its receptor.Alternatively, a binding protein may target the regions responsible forcytokine stabilization, thereby designating the protein for degradation.The methods of visualizing and modeling epitope recognition are known toone skilled in the art (US 20090311253).

“Pharmacokinetics” refers to the process by which a drug is absorbed,distributed, metabolized, and excreted by an organism. To generate amultivalent binding protein molecule with a desired pharmacokineticprofile, parent monoclonal antibodies with similarly desiredpharmacokinetic profiles are selected. The PK profiles of the selectedparental monoclonal antibodies can be easily determined in rodents usingmethods known to one skilled in the art (US 20090311253).

“Bioavailability” refers to the amount of active drug that reaches itstarget following administration. Bioavailability is function of severalof the previously described properties, including stability, solubility,immunogenicity and pharmacokinetics, and can be assessed using methodsknown to one skilled in the art (US 20090311253).

The term “surface plasmon resonance” means an optical phenomenon thatallows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore® system (BIAcore International AB,a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson et al. (1993) Ann. Biol. Clin.51:19-26. The term “K_(on)” means the on rate constant for associationof a binding protein (e.g., an antibody or DVD-Ig protein) to theantigen to form the, e.g., DVD-Ig/antigen complex. The term “K_(on)”also means “association rate constant”, or “ka”, as is usedinterchangeably herein. This value indicating the binding rate of abinding protein to its target antigen or the rate of complex formationbetween a binding protein, e.g., an antibody, and antigen also is shownby the equation below:

Antibody (“Ab”)+Antigen (“Ag”)→>Ab−Ag

The term “K_(off)” means the off rate constant for dissociation, or“dissociation rate constant”, of a binding protein (e.g., an antibody orDVD-Ig protein) from the, e.g., DVD-Ig protein/antigen complex as isknown in the art. This value indicates the dissociation rate of abinding protein, e.g., an antibody, from its target antigen orseparation of Ab−Ag complex over time into free antibody and antigen asshown by the equation below:

Ab+Ag←Ab−Ag

The terms “K_(d)” and “equilibrium dissociation constant” means thevalue obtained in a titration measurement at equilibrium, or by dividingthe dissociation rate constant (K_(off)) by the association rateconstant (K_(on)). The association rate constant, the dissociation rateconstant and the equilibrium dissociation constant, are used torepresent the binding affinity of a binding protein (e.g., an antibodyor DVD-Ig protein) to an antigen. Methods for determining associationand dissociation rate constants are well known in the art. Usingfluorescence-based techniques offers high sensitivity and the ability toexamine samples in physiological buffers at equilibrium. Otherexperimental approaches and instruments such as a BIAcore® (biomolecularinteraction analysis) assay, can be used (e.g., instrument availablefrom BIAcore International AB, a GE Healthcare company, Uppsala,Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay,available from Sapidyne Instruments (Boise, Id.), can also be used.

The term “variant” means a polypeptide that differs from a givenpolypeptide in amino acid sequence by the addition (e.g., insertion),deletion, or conservative substitution of amino acids, but that retainsthe biological activity of the given polypeptide (e.g., a variant IL-17antibody can compete with anti-IL-17 antibody for binding to IL-17). Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al. (1982)J. Mol. Biol. 157: 105-132). The hydropathic index of an amino acid isbased on a consideration of its hydrophobicity and charge. It is knownin the art that amino acids of similar hydropathic indexes in a proteincan be substituted and the protein still retains protein function. Inone aspect, amino acids having hydropathic indexes of ±2 aresubstituted. The hydrophilicity of amino acids also can be used toreveal substitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a peptide permits calculation of the greatest local averagehydrophilicity of that peptide, a useful measure that has been reportedto correlate well with antigenicity and immunogenicity (see, e.g., U.S.Pat. No. 4,554,101). Substitution of amino acids having similarhydrophilicity values can result in peptides retaining biologicalactivity, for example immunogenicity, as is understood in the art. Inone aspect, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties. The term “variant” also includes polypeptide orfragment thereof that has been differentially processed, such as byproteolysis, phosphorylation, or other post-translational modification,yet retains its biological activity or antigen reactivity, e.g., theability to bind to IL-17. The term “variant” encompasses fragments of avariant unless otherwise defined. A variant may be 99%, 98%, 97%, 96%,95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%,81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the wildtype sequence.

I. Generation of Binding Proteins

Binding proteins capable of binding IL-13 and/or IL-17 and methods ofmaking the same are provided. The binding protein can be generated usingvarious techniques. Expression vectors, host cell and methods ofgenerating the binding protein are provided and are well known in theart.

A. Generation of Parent Monoclonal Antibodies

The variable domains of the DVD binding protein can be obtained fromparent antibodies, including polyclonal Abs and mAbs capable of bindingantigens of interest. These antibodies may be naturally occurring or maybe generated by recombinant technology. The person of ordinary skill inthe art is well familiar with many methods for producing antibodies,including, but not limited to using hybridoma techniques, selectedlymphocyte antibody method (SLAM), use of a phage, yeast, or RNA-proteinfusion display or other library, immunizing a non-human animalcomprising at least some of the human immunoglobulin locus, andpreparation of chimeric, CDR-grafted, and humanized antibodies. See,e.g., US Patent Publication No. 20090311253 A1. Variable domains mayalso be prepared using affinity maturation techniques.

B. Criteria for Selecting Parent Monoclonal Antibodies

An embodiment is provided comprising selecting parent antibodies with atleast one or more properties desired in the DVD binding proteinmolecule. In an embodiment, the desired property is one or more antibodyparameters, such as, for example, antigen specificity, affinity toantigen, potency, biological function, epitope recognition, stability,solubility, production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, or orthologous antigenbinding. See, e.g., US Patent Publication No. 20090311253.

C. Construction of Binding Protein Molecules

The binding protein may be designed such that two different light chainvariable domains (VL) from the two different parent monoclonalantibodies are linked in tandem directly or via a linker by recombinantDNA techniques, followed by the light chain constant domain CL.Similarly, the heavy chain comprises two different heavy chain variabledomains (VH) linked in tandem, directly or via a linker, followed by theconstant domain CH1 and Fc region (FIG. 1).

The variable domains can be obtained using recombinant DNA techniquesfrom parent antibodies generated by any one of the methods describedherein. In an embodiment, the variable domain is a murine heavy or lightchain variable domain. In another embodiment, the variable domain is aCDR grafted or a humanized variable heavy or light chain domain. In anembodiment, the variable domain is a human heavy or light chain variabledomain.

The linker sequence may be a single amino acid or a polypeptidesequence. In an embodiment, the choice of linker sequences is based oncrystal structure analysis of several Fab molecules. There is a naturalflexible linkage between the variable domain and the CH1/CL constantdomain in Fab or antibody molecular structure. This natural linkagecomprises approximately 10-12 amino acid residues, contributed by 4-6residues from the C-terminus of a V domain and 4-6 residues from theN-terminus of a CL/CH1 domain. DVD binding proteins were generated usingN-terminal 5-6 amino acid residues, or 11-12 amino acid residues, of CLor CH1 as a linker in the light chain and heavy chains, respectively.The N-terminal residues of CL or CH1 domains, particularly the first 5-6amino acid residues, can adopt a loop conformation without strongsecondary structures, and therefore can act as flexible linkers betweenthe two variable domains. The N-terminal residues of CL or CH1 domainsare natural extension of the variable domains, as they are part of theIg sequences, and therefore their use minimizes to a large extent anyimmunogenicity potentially arising from the linkers and junctions.

In a further embodiment, of any of the heavy chain, light chain, twochain, or four chain embodiments, includes at least one linkercomprising AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ IDNO: 2); AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQID NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS(SEQ ID NO: 8); RADAAAA(G₄S)₄ (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ IDNO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15);QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP(SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20);ASTKGP (SEQ ID NO: 21); ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS(SEQ ID NO: 23); GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQID NO: 25); or GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP(SEQ ID NO: 27); ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28); or G/Sbased sequences (e.g., G4S repeats; SEQ ID NO: 29). In an embodiment, X2is an Fc region. In another embodiment, X2 is a variant Fc region.

Other linker sequences may include any sequence of any length of aCL/CH1 domain but not all residues of a CL/CH1 domain; for example thefirst 5-12 amino acid residues of a CL/CH1 domain; the light chainlinkers can be from CK or CA; and the heavy chain linkers can be derivedfrom CH1 of any isotype, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε,and Cμ. Linker sequences may also be derived from other proteins such asIg-like proteins (e.g., TCR, FcR, KIR); G/S based sequences (e.g., G4Srepeats; SEQ ID NO: 29); hinge region-derived sequences; and othernatural sequences from other proteins.

In an embodiment, a constant domain is linked to the two linked variabledomains using recombinant DNA techniques. In an embodiment, a sequencecomprising linked heavy chain variable domains is linked to a heavychain constant domain and a sequence comprising linked light chainvariable domains is linked to a light chain constant domain. In anembodiment, the constant domains are human heavy chain constant domainsand human light chain constant domains respectively. In an embodiment,the DVD heavy chain is further linked to an Fc region. The Fc region maybe a native sequence Fc region or a variant Fc region. In anotherembodiment, the Fc region is a human Fc region. In another embodiment,the Fc region includes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM,IgE, or IgD.

In another embodiment, two heavy chain DVD polypeptides and two lightchain DVD polypeptides are combined to form a DVD binding protein.Tables 1A-1C list amino acid sequences of VH and VL regions of exemplaryantibodies useful for treating disease. In an embodiment, a DVD-Igprotein comprising at least two of the VH and/or VL regions listed inTables 1A-1C, in any orientation, is provided. In some embodiments, VD1and VD2 are independently chosen. Therefore, in some embodiments, VD1and VD2 comprise the same SEQ ID NO and, in other embodiments, VD1 andVD2 comprise different SEQ ID NOS. The VH and VL domain sequencesprovided below comprise complementarity determining regions (CDRs) andframework sequences that are either known in the art or readilydiscernible using methods known in the art. In some embodiments, one ormore of these CDRs and/or framework sequences are replaced, without lossof function, by other CDRs and/or framework sequences from bindingproteins that are known in the art to bind to the same antigen.

TABLE 1A List of Amino Acid Sequences of VH and VL Regions of Antibodies forGenerating Binding Proteins, Including Multivalent Binding Proteins(CDR sequences in bold) SEQ ABT ID Unique Sequence No. ID Protein region1234567890123456789012345678901234567890 30 AB397VH VH-IL13 (seq. 1)EVTLRESGPGLVKPTQTLTLTCTLYGFSLSTSDMGVDWIRQPPGKGLEWLAHIWWDDVKRYNPALKSRLTISKDTSKNQVVLKLTSVDPVDTATYYCARTVSSGYIYYAMDYWGQGTLVT VSS 31 AB397VL VL-IL13 (seq. 1)DIQMTQSPSSLSASVGDRVTISCRASQDIRNYLNWYQQKPGKAPKLLIFYTSKLHSGVPSRFSGSGSGTDYTLTISSLQP EDIATYYCQQGLTPPLTFGGGTKVEIKR 32AB398VH VH-IL13 (seq. 2) EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYGVSWVRQAPGQGLEWMGEIYPGNYNTYYNEKFRGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARWRTSYFSDYGYFDYWGQGTTVT VSS 33 AB398VL VL-IL13 (seq. 2)DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSHGNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIKR 34 AB399VH VH-IL13 (seq. 3)EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPKGGSNIYNEKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARLDYFGDSFDLWGQGTTVTVSS 35 AB399VL VL-IL13 (seq. 3)DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLNWYQQKPGKAPKLLIYYASNLEVGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCQQDNRFPYTFGGGTKVEIKR 36AB273VH VH-IL17 (seq. 1) EVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEIHWVRQAPGQGLEWMGVNDPESGGTFYNQKFDGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRYSKWDSFDGMDYWGQGTTVTVS S 37 AB273VL VL-IL17 (seq. 1)DIQMTQSPSSLSASVGDRVTITCRASSGIISYIDWFQQKPGKAPKRLIYATFDLASGVPSRFSGSGSGTDYTLTISSLQP EDFATYYCRQVGSYPETFGQGTKLEIKR 38AB274VH VH-IL17 (seq. 2) EVQLVQSGAEVKKPGSSVKVSCKASGGSFGGYGIGWVRQAPGQGLEWMGGITPFFGFADYAQKFQGRVTITADESTTTAYMELSGLTSDDTAVYYCARDPNEFWNGYYSTHDFDSWGQGT TVTVSS 39 AB274VLVL-IL17 (seq. 2) EIVLTQSPDFQSVTPKEKVTITCRASQDIGSELHWYQQKPDQPPKLLIKYASHSTSGVPSRFSGSGSGTDFTLTINGLEA EDAGTYYCHQTDSLPYTFGPGTKVDIKR 40AB275VH VH-IL17 (seq. 3) EVQLVQSGAEVKKPGESVKISCKASGGSFRSYGISWVRQAPGQGLEWMGGITHFFGITDYAQKFQGRVTITADESTTTAYMELSGLTSDDTAVYYCAREPNDFWNGYYDTHDFDSWGQGT TVTVSS 41 AB275VLVL-IL17 (seq. 3) EIVLTQSPDFQSVTPKEKVTITCRASQNIGSELHWYQQKPDQSPKLLIKYASHSISGVPSRFSGSGSGTDFTLTINGLEA EDAATYYCHQSDTLPHTFGQGTKVDIKR

TABLE 1B  Individual IL-17 VH Sequences from Affinity Matured Clones forGenerating Binding Proteins, Including Multivalent Binding ProteinsIL-17 VH Sequences SEQ ID Sequence No. Protein region123456789012345678901234567890 42 h10f7VH.EVQLVQSGAEVKKPGSSVKVSCKASGYTFT 1a.g1m DYEIHWVRQAPGQGLEWIGVNDPESGGTFY VHNQKFDGRATLTADKSTSTAYMELSSLRSED TAVYYCTRYYRYESFYGMDYWGQGTTVTVS S h10f7VH.CDR-H1 Residues 31- DYEIH 1a.g1m 35 of SEQ ID VH NO.: 42 h10f7VH. CDR-H2Residues 50- VNDPESGGTFYNQKFDG 1a.g1m 66 of SEQ ID VH NO.: 42 h10f7VH.CDR-H3 Residues 99- YYRYESFYGMDY 1a.g1m 110 of SEQ VH ID NO.: 42 43J439M1S3 EVQLVQSGAEVKKPGSSVKVSCKASGYTFD R5#10DYEIHWVRQAPGQGLEWMGVNDPESGGTFY VH NQKFDGRATLTADKSTSTAYMELSSLRSEDTAVYYCTRYDKWDSFYGMDYWGQGTTVTVS S J439M1S3 CDR-H1 Residues 31- DYEIHR5#10 35 of SEQ ID VH NO.: 43 J439M1S3 CDR-H2 Residues 50-VNDPESGGTFYNQKFDG R5#10 66 of SEQ ID VH NO.: 43 J439M1S3 CDR-H3Residues 99- YDKWDSFYGMDY R5#10 110 of SEQ VH ID NO.: 43 44 J439M1S3EVQLVQSGAEVKKPGSSVKVSCKASGYTFT R5#11 DYEIHWVRQAPGQGLEWMGVNDPESGGTFY VHNQKFDGRVTLTADESTSTAYMELSSLRSED TAVYYCTRYSKWDSFDGMDYWGQGTTVTVS S J439M1S3CDR-H1 Residues 31- DYEIH R5#11 35 of SEQ ID VH NO.: 44 J439M1S3 CDR-H2Residues 50- VNDPESGGTFYNQKFDG R5#11 66 of SEQ ID VH NO.: 44 J439M1S3CDR-H3 Residues 99- YSKWDSFDGMDY R5#11 110 of SEQ VH ID NO.: 44 45J439M1S2 EVQLVQSGAEVKKPGSSVKVSCKASGYTFT (H)3 #A6DYEIHWVRQAPGQGLEWIGVNDPDSGGTLY VH NQKFDGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRYDKWYSFEGMDIWGQGTTVTVS S 439M1S2 CDR-H1 Residues 31- DYEIH(H)3 #A6 35 of SEQ ID VH NO.: 45 439M1S2 CDR-H2 Residues 50-VNDPDSGGTLYNQKFDG (H)3 #A6 66 of SEQ ID VH NO.: 45 439M1S2 CDR-H3Residues 99- YDKWYSFEGNDI (H)3 #A6 110 of SEQ VH ID NO.: 45 46 J439M1S2EVQLVQSGAEVKKPGSSVKVSCKASGYTFT (H)3 DYEIHWVRQAPGQGLEWMGVNDPESGGTFY #A11NQKFDGRVTLSADESTSTAYMELSSLRSED VH TAVYYCTRYDKYWSFEGMDYWGQGTTVTVS SJ439M1S2 CDR-H1 Residues 31- DYEIH (H)3 35 of SEQ ID #A11 NO.: 46 VHJ439M1S2 CDR-H2 Residues 50- VNDPESGGTFYNQKFDG (H)3 66 of SEQ ID  #A11NO.: 46 VH J439M1S2 CDR-H3 Residues 99- YDKYWSFEGNDY (H)3 110 of SEQ #A11 ID NO.: 46 VH 47 J439M1S2 EVQLVQSGAEVKKPGSSVKVSCKASGYTFS (H)3DYEIHWVRQAPGQGLEWMGVNDPESGGTFY #A16 NQKFDGRVTLTADESTSTAYMELSSLRSED VHTAVYYCTRYDKWYSFEGMDIWGQGTTVTVS S J439M1S2 CDR-H1 Residues 31- DYEIH (H)335 of SEQ ID #A16 NO.: 47 VH J439M1S2 CDR-H2 Residues 50-VNDPESGGTFYNQKFDG (H)3 66 of SEQ ID #A16 NO.: 47 VH J439M1S2 CDR-H3Residues 99- DKWYSFEGMDI (H)3 110 of SEQ #A16 ID NO.: 47 VH 48 J439M1S2EVQLVQSGAEVKKPGSSVKVSCKASGYTFS (H)3 DYEIHWVRQAPGQGLEWMGVNDPESGGTFY #B13NQKFDGRVTLTADESTSTAYMELSSLRSED VH TAVYYCTRYDKYWSFEGMDYWGQGTTVTVS SJ439M1S2 CDR-H1  Residues 31- DYEIH (H)3 35 of SEQ ID #B13 NO.: 48 VHJ439M1S2 (H)3 CDR-H2 Residues 50-  VNDPESGGTFYNQKFDG #B13 66 of SEQ IDVH NO.: 48 J439M1S2 CDR-H3 Residues 99- DKYWSFEGMDY (H)3 110 of SEQ #B13ID NO.: 48 VH 49 J439M1S2 EVQLVQSGAEVKKPGSSVKVSCKASGYTFT (H)3DYEIHWVRQAPGQGLEWMGVNDPESGGTFY #B20 NQKFDGRVTLTADESTSTAYMELSSLRSED VHTAVYYCTRYDKWYSFEGMDIWGQGTTVTVS S J439M1S2 CDR-H1  Residues 31- DYEIH(H)3 35 of SEQ ID #B20 NO.: 49 VH J439M1S2 CDR-H2  Residues 50-VNDPESGGTFYNQKFDG (H)3 66 of SEQ ID  #B20 NO.: 49 VH J439M1S2 CDR-H3Residues 99- DKWYSFEGMDI (H)3 110 of SEQ #B20 ID NO.: 49 VH

TABLE 1C Individual IL-17 VL Sequences from Affinity Matured Clones forGenerating Binding Proteins, Including Multivalent BindingProteins IL-17 VL Sequences SEQ ID Sequence No. Protein region123456789012345678901234567890 50 J439M1S3DIQMTQSPSSLSASVGDRVTITCSASSGSI R5#10 SYIDWFQQKPGKAPKRLIYATFELASGVPS VLRFSGSGSGTDFTLTISSLQPEDFATYYCHQ LGSYPDTFGQGTKLEIK J439M1S3 CDR-L1Residues SASSGSISYID R5#10 24-34 of VL SEQ ID NO.: 50 J439M1S3 CDR-L2Residues ATFELAS R5#10 50-56 of VL SEQ ID NO.: 50 J439M1S3 CDR-L3Residues HQLGSYPDT R5#10 89-97 of VL SEQ ID NO.: 50 51 J439M1S3DIQMTQSPSSLSASVGDRVTITCRASSGII R5#11 SYIDWFQQKPGKAPKRLIYATFDLASGVPS VLRFSGSGSGTDYTLTISSLQPEDFATYYCRQ VGSYPETFGQGTKLEIK J439M1S3 CDR-L1Residues RASSGIISYID R5#11 24-34 of VL SEQ ID NO.: 51 J439M1S3 CDR-L2Residues ATFDLAS R5#11 50-56 of VL SEQ ID NO.: 51 J439M1S3 CDR-L3Residues RQVGSYPET R5#11 89-97 of  VL SEQ ID NO.: 51 52 J427M2S3DIQMTQSPSSLSASVGDRVTITCSASSGII #12 SSIDWFQQKPGKAPKRLIYATFALQSGVPS VLRFSGSGSGTDFTLTISSLQPEDFATYYCSQ MSSYPHTFGQGTKLEIK J427M2S3 CDR-L1Residues SASSGIISSID #12 24-34 of VL SEQ ID NO.: 52 J427M2S3 CDR-L2Residues ATFALQS #12 50-56 of VL SEQ ID NO.: 52 J427M2S3 CDR-L3 ResiduesSQMSSYPHT #12 89-97 of VL SEQ ID NO.: 52 53 J427M2S3 ResiduesDIQMTQSPSSLSASVGDRVTITCSASSDIS #27 24-34 ofSYLNWFQQKPGKSPKRLIYRTSELQSGVPS VL SEQ ID RFSGSGSGTDFTLTISSLQPEDFATYYCQQNO.: 53 WSSYPWTFGQGTKLEIK J427M2S3 CDR-L1 Residues SASSDISSYLN #2750-56 of VL SEQ ID NO.: 53 J427M2S3 CDR-L2 Residues RTSELQS #27 89-97 ofVL SEQ ID NO.: 53 J427M2S3 CDR-L3 QQWSSYPWT #27 VL 54 J439M1S2DIQMTQSPSSLSASVGDRVTITCSASQGIR (H)3#A6 SYIDWFQQKPGKSPKRLIYATFDLASGVPS VLRFSGSGSGTDYTLTISSLQPEDFATYYCRQ VGNYPGTFGQGTKLEIK J439M1S2 CDR-L1Residues SASQGIRSYID (H)3#A6 24-34 of VL SEQ ID NO.: 54 J439M1S2 CDR-L2Residues ATFDLAS (H)3#A6 50-56 of VI SEQ ID NO.: 54 J439M1S2 CDR-L3Residues RQVGNYPGT (H)3#A6 89-97 of VL SEQ ID NO.: 54

Detailed description of specific DVD binding proteins capable of bindingspecific targets, and methods of making the same, is provided in theExamples section below.

D. Production of Binding Proteins

The binding proteins provided herein may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the DVD heavy and DVD light chainsis (are) transfected into a host cell by standard techniques. Althoughit is possible to express the DVD binding proteins provided herein ineither prokaryotic or eukaryotic host cells, DVD binding proteins areexpressed in eukaryotic cells, for example, mammalian host cells,because such eukaryotic cells (and in particular mammalian cells) aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active DVD binding protein.

In an exemplary system for recombinant expression of DVD proteins, arecombinant expression vector encoding both the DVD heavy chain and theDVD light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the DVD heavy and light chain genes are each operatively linkedto CMV enhancer/AdMLP promoter regulatory elements to drive high levelsof transcription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the DVD heavy and light chains andintact DVD protein is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the DVD protein from the culturemedium. A method of synthesizing a DVD protein provided herein byculturing a host cell provided herein in a suitable culture medium untila DVD protein is synthesized is also provided. The method can furthercomprise isolating the DVD protein from the culture medium.

An important feature of DVD binding protein is that it can be producedand purified in a similar way as a conventional antibody. The productionof DVD binding protein results in a homogeneous, single major productwith desired dual-specific activity, without the need for sequencemodification of the constant region or chemical modifications. Otherpreviously described methods to generate “bi-specific”,“multi-specific”, and “multi-specific multivalent” full length bindingproteins can lead to the intracellular or secreted production of amixture of assembled inactive, mono-specific, multi-specific,multivalent, full length binding proteins, and multivalent full lengthbinding proteins with a combination of different binding sites.

Surprisingly, the design of the “dual-specific multivalent full lengthbinding proteins” provided herein leads to a dual variable domain lightchain and a dual variable domain heavy chain that assemble primarily tothe desired “dual-specific multivalent full length binding proteins”.

At least 50%, at least 75% and at least 90% of the assembled, andexpressed dual variable domain immunoglobulin molecules are the desireddual-specific tetravalent protein, and therefore possess enhancedcommercial utility. Thus, a method to express a dual variable domainlight chain and a dual variable domain heavy chain in a single cellleading to a single primary product of a “dual-specific tetravalent fulllength binding protein” is provided.

Methods of expressing a dual variable domain light chain and a dualvariable domain heavy chain in a single cell leading to a “primaryproduct” of a “dual-specific tetravalent full length binding protein”,where the “primary product” is more than 50%, such as more than 75% andmore than 90%, of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain are provided.

II. Uses of Binding Proteins

Given their ability to bind to two or more antigens the binding proteinsprovided herein can be used to detect the antigens (e.g., in abiological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), aradioimmunoassay (RIA), or tissue immunohistochemistry. The bindingprotein is directly or indirectly labeled with a detectable substance tofacilitate detection of the bound or unbound antibody. Suitabledetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anexample of a luminescent material is luminol and examples of suitableradioactive materials include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I,¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm.

In an embodiment, the binding proteins provided herein are capable ofneutralizing the activity of their antigen targets both in vitro and invivo. Accordingly, such binding proteins can be used to inhibit antigenactivity, e.g., in a cell culture containing the antigens, in humansubjects or in other mammalian subjects having the antigens with which abinding protein provided herein cross-reacts. In another embodiment, amethod for reducing antigen activity in a subject suffering from adisease or disorder in which the antigen activity is detrimental isprovided. A binding protein provided herein can be administered to ahuman subject for therapeutic purposes.

The term “a disorder in which antigen activity is detrimental” isintended to include diseases and other disorders in which the presenceof the antigen in a subject suffering from the disorder has been shownto be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which antigenactivity is detrimental is a disorder in which reduction of antigenactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of the antigen in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofantigen in serum, plasma, synovial fluid, etc., of the subject).Non-limiting examples of disorders that can be treated with the bindingproteins provided herein include those disorders discussed below and inthe section pertaining to pharmaceutical compositions comprising thebinding proteins.

DVD binding proteins are useful as therapeutic agents to simultaneouslyblock two different targets to enhance efficacy/safety and/or increasepatient coverage.

Additionally, DVD binding proteins provided herein can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand an intracellular molecule), delivering to inside brain (targetingtransferrin receptor and a CNS disease mediator for crossing theblood-brain barrier). DVD binding protein can also serve as a carrierprotein to deliver an antigen to a specific location via binding to anon-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, DVD binding protein can bedesigned to either be physically linked to medical devices implantedinto patients or target these medical devices (see Burke et al. (2006)Advanced Drug Deliv. Rev. 58(3): 437-446; Hildebrand et al. (2006)Surface and Coatings Technol. 200(22-23): 6318-6324; Drug/devicecombinations for local drug therapies and infection prophylaxis, Wu(2006) Biomaterials 27(11):2450-2467; Mediation of the cytokine networkin the implantation of orthopedic devices, Marques (2005) BiodegradableSystems in Tissue Engineer. Regen. Med. 377-397). Briefly, directingappropriate types of cell to the site of medical implant may promotehealing and restoring normal tissue function. Alternatively, inhibitionof mediators (including but not limited to cytokines), released upondevice implantation by a DVD coupled to or target to a device is alsoprovided.

A. Use of Binding Proteins in Various Diseases

Binding protein molecules provided herein are useful as therapeuticmolecules to treat various diseases, e.g., wherein the targets that arerecognized by the binding proteins are detrimental. Such bindingproteins may bind one or more targets involved in a specific disease.Inhibition of IL-13 and/or IL-17 has also been shown to enhanceanti-viral vaccines in animal models and may be beneficial in thetreatment of HIV and other infectious diseases, for example, the humanrhinovirus, other enteroviruses, coronavirus, herpes viruses, influenzavirus, parainfluenza virus, respiratory syncytial virus or adenovirus.

Without limiting the disclosure, further information on certain diseaseconditions is provided.

1. Human Autoimmune and Inflammatory Response

IL-13 and/or IL-17 have been implicated in general autoimmune andinflammatory responses, including, for example, asthma, allergies,allergic lung disease, allergic rhinitis, atopic dermatitis, chronicobstructive pulmonary disease (COPD), fibrosis, cystic fibrosis (CF),fibrotic lung disease, idiopathic pulmonary fibrosis, liver fibrosis,lupus, hepatitis B-related liver diseases and fibrosis, sepsis, systemiclupus erythematosus (SLE), glomerulonephritis, inflammatory skindiseases, psoriasis, diabetes, insulin dependent diabetes mellitus,inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn'sdisease (CD), rheumatoid arthritis (RA), osteoarthritis (OA), multiplesclerosis (MS), graft-versus-host disease (GVHD), transplant rejection,ischemic heart disease (IHD), celiac disease, contact hypersensitivity,alcoholic liver disease, Behcet's disease, atherosclerotic vasculardisease, occular surface inflammatory diseases, or Lyme disease.

The binding proteins provided herein can be used to treat neurologicaldisorders. In an embodiment, the binding proteins provided herein orantigen-binding portions thereof, are used to treat neurodegenerativediseases, and conditions involving neuronal regeneration and spinal cordinjury.

2. Asthma

Allergic asthma is characterized by the presence of eosinophilia, gobletcell metaplasia, epithelial cell alterations, airway hyperreactivity(AHR), and Th2 and Th1 cytokine expression, as well as elevated serumIgE levels. Corticosteroids are the most important anti-inflammatorytreatment for asthma today, however their mechanism of action isnon-specific and safety concerns exist, especially in the juvenilepatient population. The development of more specific and targetedtherapies is therefore warranted. There is increasing evidence thatIL-13 in mice mimics many of the features of asthma, including AHR,mucus hypersecretion and airway fibrosis, independently of eosinophilicinflammation (Finotto et al. (2005) Internat. Immunol. 17(8): 993-1007;Padilla et al. (2005) J. Immunol. 174(12): 8097-8105).

IL-13 has been implicated as having a pivotal role in causingpathological responses associated with asthma. The development ofanti-IL-13 mAb therapy to reduce the effects of IL-13 in the lung is anexciting new approach that offers considerable promise as a noveltreatment for asthma. However other mediators of differentialimmunological pathways are also involved in asthma pathogenesis, andblocking these mediators, in addition to IL-13, may offer additionaltherapeutic benefit. Such target pairs include, but are not limited to,IL-13 and a pro-inflammatory cytokine, such as IL-17. There is growingevidence that IL-17 is involved in the pathogenesis of asthma. IL-17induces the neutrophils into the airways and also enhances T-helper 2(Th2) cell-mediated eosinophilic airway inflammation in asthma. Recentstudies have demonstrated that inhibitors and other diverse regulatorsof IL-17 reduce antigen-induced airway inflammation, bronchialhyperresponsiveness, and Th2 cytokine levels in animal models of asthma(for a review see Park and Lee (2010) Respiratory Res., 11:78).

Animal models such as an OVA-induced asthma mouse model, where bothinflammation and AHR can be assessed, are known in the art and may beused to determine the ability of various binding protein molecules totreat asthma. Animal models for studying asthma are disclosed inCoffman, et al. (2005) J. Exp. Med. 201(12):1875-1879; Lloyd et al.(2001) Adv. Immunol. 77: 263-295; Boyce et al. (2005) J. Exp. Med.201(12):1869-1873; and Snibson et al. (2005) J. Brit. Soc. Allergy Clin.Immunol. 35(2):146-52. In addition to routine safety assessments ofthese target pairs specific tests for the degree of immunosuppressionmay be warranted and helpful in selecting the best target pairs (seeLuster et al. (1994) Toxicol. 92(1-3):229-43; Descotes et al. (1992)Dev. Biol. Standard. 77:99-102; Hart et al. (2001) J. Allergy Clin.Immunol. 108(2):250-257).

3. Rheumatoid Arthritis

Rheumatoid arthritis (RA), a systemic disease, is characterized by achronic inflammatory reaction in the synovium of joints and isassociated with degeneration of cartilage and erosion of juxta-articularbone. Many pro-inflammatory cytokines, chemokines, and growth factorsare expressed in diseased joints. Recent studies indicate that theinvolvement of T cells in RA is mediated to a significant extent byIL-17. Animal studies have shown that markedly increased IL-17expression was detected in mice that develop articular lesionsresembling human RA. Beneficial effects of IL-17 blockade were alsoobserved various animal models of the disease (for a review see Witowskiet al. (2004) Cell. Mol. Life. Sci. 61: 567-579). Whether a bindingprotein molecule will be useful for the treatment of rheumatoidarthritis can be assessed using pre-clinical animal RA models such asthe collagen-induced arthritis mouse model. Other useful models are alsowell known in the art (see Brand (2005) Comp. Med. 55(2):114-22). Basedon the cross-reactivity of the parental antibodies for human and mouseorthologues (e.g., reactivity for human and mouse TNF, human and mouseIL-15, etc.) validation studies in the mouse CIA model may be conductedwith “matched surrogate antibody” derived binding protein molecules;briefly, a binding protein based on two (or more) mouse target specificantibodies may be matched to the extent possible to the characteristicsof the parental human or humanized antibodies used for human bindingprotein construction (e.g., similar affinity, similar neutralizationpotency, similar half-life, etc.).

4. Systemic Lupus Erythematosus (SLE)

The immunopathogenic hallmark of SLE is the polyclonal B cellactivation, which leads to hyperglobulinemia, autoantibody productionand immune complex formation. Significant increased levels of IL-13 andIL-17 have been detected in patients with systemic lupus erythematosus(Morimoto et al. (2001) Autoimmunity, 34(1):19-25; Wong et al. (2008)Clin Immunol. 127(3):385-93). IL-17 represents an important cytokine inthe pathogenesis of SLE. Increased IL-17 production has been shown inpatients with SLE as well as in animals with lupus-like diseases. Animalmodels have demonstrated that blockade of IL-17 decreases lupusmanifestations (for a review see Nalbandian et al. (2009) 157(2):209-215). Based on the cross-reactivity of the parental antibodies forhuman and mouse othologues (e.g., reactivity for human and mouse CD20,human and mouse interferon alpha, etc.) validation studies in a mouselupus model may be conducted with “matched surrogate antibody” derivedbinding protein molecules. Briefly, a binding protein based two (ormore) mouse target specific antibodies may be matched to the extentpossible to the characteristics of the parental human or humanizedantibodies used for human binding protein construction (e.g., similaraffinity, similar neutralization potency, similar half-life, etc.).

5. Multiple Sclerosis

Multiple sclerosis (MS) is a complex human autoimmune-type disease witha predominantly unknown etiology. Immunologic destruction of myelinbasic protein (MBP) throughout the nervous system is the major pathologyof multiple sclerosis. Of major consideration are immunologicalmechanisms that contribute to the development of autoimmunity. Inparticular, antigen expression, cytokine and leukocyte interactions, andregulatory T-cells, which help balance/modulate other T-cells such asTh1 and Th2 cells, are important areas for therapeutic targetidentification. In MS, increased expression of IL-17 has been detectedboth in brain lesions and in mononuclear cells isolated from blood andcerebrospinal fluid. IL-17-producing cells are extremely enriched inactive MS lesions, suggesting that neutralization of this cytokine hasthe potential of being beneficial (for a review see Witowski et al.(2004) Cell. Mol. Life. Sci. 61: 567-579).

Several animal models for assessing the usefulness of the bindingproteins to treat MS are known in the art (see Steinman et al. (2005)Trends Immunol. 26(11):565-71; Lublin et al. (1985) Springer Semin.Immunopathol.8(3):197-208; Genain et al. (1997) J. Mol. Med.75(3):187-97; Tuohy et al. (1999) J. Exp. Med. 189(7):1033-42; Owens etal. (1995) Neurol. Clin. 13(1):51-73; and Hart et al. (2005) J. Immunol.175(7):4761-8.) Based on the cross-reactivity of the parental antibodiesfor human and animal species othologues validation studies in the mouseEAE model may be conducted with “matched surrogate antibody” derivedbinding protein molecules. Briefly, a binding protein based on two (ormore) mouse target specific antibodies may be matched to the extentpossible to the characteristics of the parental human or humanizedantibodies used for human binding protein construction (e.g., similaraffinity, similar neutralization potency, similar half-life, etc.). Thesame concept applies to animal models in other non-rodent species, wherea “matched surrogate antibody” derived binding protein would be selectedfor the anticipated pharmacology and possibly safety studies. Inaddition to routine safety assessments of these target pairs specifictests for the degree of immunosuppression may be warranted and helpfulin selecting the best target pairs (see Luster et al. (1994) Toxicol.92(1-3): 229-43; Descotes et al. (1992) Devel. Biol. Standard. 77:99-102; Jones (2000) IDrugs 3(4):442-6).

6. Sepsis

Overwhelming inflammatory and immune responses are essential features ofseptic shock and play a central part in the pathogenesis of tissuedamage, multiple organ failure, and death induced by sepsis. Cytokineshave been shown to be mediators of septic shock. These cytokines have adirect toxic effect on tissues; they also activate phospholipase A2.These and other effects lead to increased concentrations ofplatelet-activating factor, promotion of nitric oxide synthase activity,promotion of tissue infiltration by neutrophils, and promotion ofneutrophil activity. IL-17 levels and clinical prognosis of sepsis havebeen shown to be negatively correlated. Neutralization of IL-17A cansignificantly improve the survival rate of patients with sepsis (seeFlierl et al. (2008) FASEB J. 22: 2198-2205).

One embodiment pertains to binding proteins capable of binding one ormore targets involved in sepsis, such as, for example IL-13 and IL-17.The efficacy of such binding proteins for treating sepsis can beassessed in preclinical animal models known in the art (see Buras et al.(2005) Nat. Rev. Drug Discov. 4(10):854-65 and Calandra et al. (2000)Nat. Med. 6(2):164-70).

7. Neurological Disorders

a. Neurodegenerative Diseases

Neurodegenerative diseases are either chronic in which case they areusually age-dependent or acute (e.g., stroke, traumatic brain injury,spinal cord injury, etc.). They are characterized by progressive loss ofneuronal functions (e.g., neuronal cell death, axon loss, neuriticdystrophy, demyelination), loss of mobility and loss of memory. Thesechronic neurodegenerative diseases represent a complex interactionbetween multiple cell types and mediators. Treatment strategies for suchdiseases are limited and mostly constitute either blocking inflammatoryprocesses with non-specific anti-inflammatory agents (e.g.,corticosteroids, COX inhibitors) or agents to prevent neuron loss and/orsynaptic functions. These treatments fail to stop disease progression.Specific therapies targeting more than one disease mediator may provideeven better therapeutic efficacy for chronic neurodegenerative diseasesthan observed with targeting a single disease mechanism (see Deane etal. (2003) Nature Med. 9:907-13; and Masliah et al. (2005) Neuron.46:857).

The binding protein molecules provided herein can bind one or moretargets involved in chronic neurodegenerative diseases such asAlzheimers. The efficacy of binding protein molecules can be validatedin pre-clinical animal models such as the transgenic mice thatover-express amyloid precursor protein or RAGE and develop Alzheimer'sdisease-like symptoms. In addition, binding protein molecules can beconstructed and tested for efficacy in the animal models and the besttherapeutic binding protein can be selected for testing in humanpatients. Binding protein molecules can also be employed for treatmentof other neurodegenerative diseases such as Parkinson's disease.

b. Neuronal Regeneration and Spinal Cord Injury

Despite an increase in knowledge of the pathologic mechanisms, spinalcord injury (SCI) is still a devastating condition and represents amedical indication characterized by a high medical need. Most spinalcord injuries are contusion or compression injuries and the primaryinjury is usually followed by secondary injury mechanisms (inflammatorymediators e.g., cytokines and chemokines) that worsen the initial injuryand result in significant enlargement of the lesion area, sometimes morethan 10-fold. IL-17 is a mediator of secondary degeneration, whichcontributes to neuroinflammation and hinders functional recovery.Studies using IL-17 KO mice have demonstrated that IL-17 contributes toneuroinflammatory responses and pain hypersensitivity followingneuropathic injury (Kim and Moalem-Taylor (2010) J. Pain. 12(3):370-83).IL-17 deficiency improves locomotor recovery and tissue sparing afterspinal cord contusion injury in mice (Hill at al. (2011) Neurosci Lett.487(3):363-7).

The efficacy of binding protein molecules can be validated inpre-clinical animal models of spinal cord injury. In addition, thesebinding protein molecules can be constructed and tested for efficacy inthe animal models and the best therapeutic binding protein can beselected for testing in human patients. In general, antibodies do notcross the blood brain barrier (BBB) in an efficient and relevant manner.However, in certain neurologic diseases, e.g., stroke, traumatic braininjury, multiple sclerosis, etc., the BBB may be compromised and allowsfor increased penetration of binding proteins and antibodies into thebrain. In other neurological conditions, where BBB leakage is notoccurring, one may employ the targeting of endogenous transport systems,including carrier-mediated transporters such as glucose and amino acidcarriers and receptor-mediated transcytosis-mediating cellstructures/receptors at the vascular endothelium of the BBB, thusenabling trans-BBB transport of the binding protein. Structures at theBBB enabling such transport include but are not limited to the insulinreceptor, transferrin receptor, LRP and RAGE. In addition, strategiesenable the use of binding proteins also as shuttles to transportpotential drugs into the CNS including low molecular weight drugs,nanoparticles and nucleic acids (Coloma et al. (2000) Pharm Res.17(3):266-74; Boado et al. (2007) Bioconjug. Chem. 18(2):447-55).

8. Oncological Disorders

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (von Mehren et al. (2003) Annu. Rev. Med.54:343-69). The use of dual-specific antibody that targets two separatetumor mediators will likely give additional benefit compared to amono-specific therapy. IL-17 has been suggested to support tumor growth,probably by stimulating angiogenesis. IL-13 plays an important role inthe regulation of anti-tumor immunity and tumor growth. Studies indicatethat IL-13 is central to a novel immunoregulatory pathway in which NKTcells suppress tumor immunosurveillance. (For a review see Kolls et al.(2003) Am. J. Respir. Cell Mol. Biol. 28: 9-11, and Terabe et al. (2004)Cancer Immunol Immunother. 53(2):79-85.)

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods provided herein include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

In an embodiment, the antibodies provided herein or antigen-bindingportions thereof, are used to treat cancer or in the prevention ofmetastases from the tumors described herein either when used alone or incombination with radiotherapy and/or other chemotherapeutic agents.

9. Gene Therapy

In a specific embodiment, nucleic acid sequences encoding a bindingprotein provided herein or another prophylactic or therapeutic agentprovided herein are administered to treat, prevent, manage, orameliorate a disorder or one or more symptoms thereof by way of genetherapy. Gene therapy refers to therapy performed by the administrationto a subject of an expressed or expressible nucleic acid. In thisembodiment, the nucleic acids produce their encoded antibody orprophylactic or therapeutic agent provided herein that mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be used inthe methods provided herein. For general reviews of the methods of genetherapy, see Goldspiel et al. (1993) Clin. Pharmacy 12:488-505; Wu andWu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan (1993) Science 260:926-932; Morgan andAnderson (1993) Ann. Rev. Biochem. 62:191-217; and May (1993) TIBTECH11(5):155-215. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993); andKriegler, Gene Transfer and Expression, A Laboratory Manual, StocktonPress, NY (1990). Detailed description of various methods of genetherapy are disclosed in US Patent Publication No. US20050042664.

III. Pharmaceutical Compositions

Pharmaceutical compositions comprising one or more binding proteins,either alone or in combination with prophylactic agents, therapeuticagents, and/or pharmaceutically acceptable carriers are provided. Thepharmaceutical compositions comprising binding proteins provided hereinare for use in, but not limited to, diagnosing, detecting, or monitoringa disorder, in preventing, treating, managing, or ameliorating adisorder or one or more symptoms thereof, and/or in research. Theformulation of pharmaceutical compositions, either alone or incombination with prophylactic agents, therapeutic agents, and/orpharmaceutically acceptable carriers, are known to one skilled in theart (US Patent Publication No. 20090311253 A1).

Methods of administering a prophylactic or therapeutic agent providedherein include, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration,mucosal administration (e.g., intranasal and oral routes) and pulmonaryadministration (e.g., aerosolized compounds administered with an inhaleror nebulizer). The formulation of pharmaceutical compositions forspecific routes of administration, and the materials and techniquesnecessary for the various methods of administration are available andknown to one skilled in the art (US Patent Publication No. 20090311253A1).

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. The term“dosage unit form” refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit formsprovided herein are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a binding protein provided hereinis 0.1-20 mg/kg, for example, 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens may be adjusted over time according tothe individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

IV. Combination Therapy

A binding protein provided herein also can also be administered with oneor more additional therapeutic agents useful in the treatment of variousdiseases, the additional agent being selected by the skilled artisan forits intended purpose. For example, the additional agent can be atherapeutic agent art-recognized as being useful to treat the disease orcondition being treated by the antibody provided herein. The combinationcan also include more than one additional agent, e.g., two or threeadditional agents.

Combination therapy agents include, but are not limited to,antineoplastic agents, radiotherapy, chemotherapy such as DNA alkylatingagents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel,docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines,adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors,5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinaseinhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g.,celecoxib), kinase inhibitors, and siRNAs.

Combinations to treat autoimmune and inflammatory diseases arenon-steroidal anti-inflammatory drug(s) also referred to as NSAIDS whichinclude drugs like ibuprofen. Other combinations are corticosteroidsincluding prednisolone; the well known side-effects of steroid use canbe reduced or even eliminated by tapering the steroid dose required whentreating patients in combination with the binding proteins providedherein. Non-limiting examples of therapeutic agents for rheumatoidarthritis with which an antibody provided herein, or antibody bindingportion thereof, can be combined include the following: cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); antibodies to orantagonists of other human cytokines or growth factors, for example,TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16,IL-18, IL-21, IL-23, interferons, EMAP-II, GM-CSF, FGF, and PDGF.Binding proteins provided herein, or antigen binding portions thereof,can be combined with antibodies to cell surface molecules such as CD2,CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86(B7.2), CD90, CTLA or their ligands including CD154 (gp39 or CD40L).

Combinations of therapeutic agents may interfere at different points inthe autoimmune and subsequent inflammatory cascade. Examples include abinding protein disclosed herein and a TNF antagonist like a chimeric,humanized or human TNF antibody, Adalimumab, (PCT Publication No. WO97/29131), CA2 (Remicade™), CDP 571, a soluble p55 or p75 TNF receptor,or derivative thereof (p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept)),a TNFα converting enzyme (TACE) inhibitor; or an IL-1 inhibitor (anInterleukin-1-converting enzyme inhibitor, IL-1RA, etc.). Othercombinations include a binding protein disclosed herein and Interleukin11. Yet another combination include key players of the autoimmuneresponse which may act parallel to, dependent on or in concert withIL-12 function; especially relevant are IL-18 antagonists including anIL-18 antibody, a soluble IL-18 receptor, or an IL-18 binding protein.It has been shown that IL-12 and IL-18 have overlapping but distinctfunctions and a combination of antagonists to both may be mosteffective. Yet another combination is a binding protein disclosed hereinand a non-depleting anti-CD4 inhibitor. Yet other combinations include abinding protein disclosed herein and an antagonist of the co-stimulatorypathway CD80 (B7.1) or CD86 (B7.2) including an antibody, a solublereceptor, or an antagonistic ligand.

The binding proteins provided herein may also be combined with an agent,such as methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine,olsalazine chloroquinine/hydroxychloroquine, pencillamine,aurothiomalate (intramuscular and oral), azathioprine, cochicine, acorticosteroid (oral, inhaled and local injection), a beta-2adrenoreceptor agonist (salbutamol, terbutaline, salmeteral), a xanthine(theophylline, aminophylline), cromoglycate, nedocromil, ketotifen,ipratropium, oxitropium, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, an NSAID, for example, ibuprofen, a corticosteroidsuch as prednisolone, a phosphodiesterase inhibitor, an adensosineagonist, an antithrombotic agent, a complement inhibitor, an adrenergicagent, an agent which interferes with signalling by proinflammatorycytokines such as TNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38 or a MAPkinase inhibitor), an IL-1β converting enzyme inhibitor, a TNFαconverting enzyme (TACE) inhibitor, a T-cell signalling inhibitor suchas a kinase inhibitor, a metalloproteinase inhibitor, sulfasalazine,azathioprine, a 6-mercaptopurine, an angiotensin converting enzymeinhibitor, a soluble cytokine receptor or derivative thereof (e.g., asoluble p55 or p75 TNF receptor or the derivative p75TNFRIgG (Enbrel™)or p55TNFRIgG (Lenercept), sIL-1RI, sIL-1RII, sIL-6R), anantiinflammatory cytokine (e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ),celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib,etanercept, infliximab, naproxen, valdecoxib, sulfasalazine,methylprednisolone, meloxicam, methylprednisolone acetate, gold sodiumthiomalate, aspirin, triamcinolone acetonide, propoxyphenenapsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac,diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodonebitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra,human recombinant, tramadol hcl, salsalate, sulindac,cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin,glucosamine sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodonehcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium,omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,Roflumilast, IC-485, CDC-801, or Mesopram. Combinations includemethotrexate or leflunomide and in moderate or severe rheumatoidarthritis cases, cyclosporine.

In one embodiment, the binding protein or antigen-binding portionthereof, is administered in combination with one of the following agentsfor the treatment of rheumatoid arthritis: a small molecule inhibitor ofKDR, a small molecule inhibitor of Tie-2; methotrexate; prednisone;celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib;etanercept; infliximab; leflunomide; naproxen; valdecoxib;sulfasalazine; methylprednisolone; ibuprofen; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; amitriptyline hcl; sulfadiazine; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15;BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;CDC-801; or mesopram.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which a binding protein provided herein can be combinedinclude the following: budenoside; epidermal growth factor; acorticosteroid; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; a lipoxygenase inhibitor;mesalamine; olsalazine; balsalazide; an antioxidant; a thromboxaneinhibitor; an IL-1 receptor antagonist; an anti-IL-1β mAb; an anti-IL-6mAb; a growth factor; an elastase inhibitor; a pyridinyl-imidazolecompound; an antibody to or antagonist of other human cytokines orgrowth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, or PDGF. Antibodiesprovided herein, or antigen binding portions thereof, can be combinedwith an antibody to a cell surface molecule such as CD2, CD3, CD4, CD8,CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands. Theantibodies provided herein, or antigen binding portions thereof, mayalso be combined with an agent, such as methotrexate, cyclosporin,FK506, rapamycin, mycophenolate mofetil, leflunomide, an NSAID, forexample, ibuprofen, a corticosteroid such as prednisolone, aphosphodiesterase inhibitor, an adenosine agonist, an antithromboticagent, a complement inhibitor, an adrenergic agent, an agent whichinterferes with signalling by proinflammatory cytokines such as TNFα orIL-1 (e.g., an IRAK, NIK, IKK, p38 or MAP kinase inhibitor), an IL-1βconverting enzyme inhibitor, a TNFα converting enzyme inhibitor, aT-cell signalling inhibitor such as a kinase inhibitor, ametalloproteinase inhibitor, sulfasalazine, azathioprine, a6-mercaptopurine, an angiotensin converting enzyme inhibitor, a solublecytokine receptor or derivative thereof (e.g., a soluble p55 or p75 TNFreceptor, sIL-1RI, sIL-1RII, sIL-6R) or an antiinflammatory cytokine(e.g., IL-4, IL-10, IL-11, IL-13 or TGFβ) or a bcl-2 inhibitor.

Examples of therapeutic agents for Crohn's disease in which a bindingprotein can be combined include the following: a TNF antagonist, forexample, an anti-TNF antibody, Adalimumab (PCT Publication No. WO97/29131; HUMIRA), CA2 (REMICADE), CDP 571, a TNFR-Ig construct,(p75TNFRIgG (ENBREL) or a p55TNFRIgG (LENERCEPT)) inhibitor or a PDE4inhibitor. Antibodies provided herein, or antigen binding portionsthereof, can be combined with a corticosteroid, for example, budenosideand dexamethasone. Binding proteins provided herein or antigen bindingportions thereof, may also be combined with an agent such assulfasalazine, 5-aminosalicylic acid and olsalazine, or an agent thatinterferes with the synthesis or action of a proinflammatory cytokinesuch as IL-1, for example, an IL-1β converting enzyme inhibitor or IL-1ra. Antibodies provided herein or antigen binding portion thereof mayalso be used with a T cell signaling inhibitor, for example, a tyrosinekinase inhibitor or an 6-mercaptopurine. Binding proteins providedherein, or antigen binding portions thereof, can be combined with IL-11.Binding proteins provided herein, or antigen binding portions thereof,can be combined with mesalamine, prednisone, azathioprine,mercaptopurine, infliximab, methylprednisolone sodium succinate,diphenoxylate/atrop sulfate, loperamide hydrochloride, methotrexate,omeprazole, folate, ciprofloxacin/dextrose-water, hydrocodonebitartrate/apap, tetracycline hydrochloride, fluocinonide,metronidazole, thimerosal/boric acid, cholestyramine/sucrose,ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidinehydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen,promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab or interferon-gamma

Non-limiting examples of therapeutic agents for multiple sclerosis withwhich binding proteins provided herein can be combined include thefollowing: a corticosteroid; prednisolone; methylprednisolone;azathioprine; cyclophosphamide; cyclosporine; methotrexate;4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen);interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (InterferonSciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferonβ1A-1F (Serono/Inhale Therapeutics), Peginterferon α 2b(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen; intravenousimmunoglobulin; clabribine; an antibody to or antagonist of other humancytokines or growth factors and their receptors, for example, TNF, LT,IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, or PDGF. Binding proteins provided herein can be combinedwith an antibody to a cell surface molecule such as CD2, CD3, CD4, CD8,CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. Binding proteins provided herein, may also be combinedwith an agent, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, an NSAID, for example, ibuprofen, acorticosteroid such as prednisolone, a phosphodiesterase inhibitor, anadensosine agonist, an antithrombotic agent, a complement inhibitor, anadrenergic agent, an agent which interferes with signalling by aproinflammatory cytokine such as TNFα or IL-1 (e.g., IRAK, NIK, IKK, p38or a MAP kinase inhibitor), an IL-1β converting enzyme inhibitor, a TACEinhibitor, a T-cell signaling inhibitor such as a kinase inhibitor, ametalloproteinase inhibitor, sulfasalazine, azathioprine, a6-mercaptopurine, an angiotensin converting enzyme inhibitor, a solublecytokine receptor or derivatives thereof (e.g., a soluble p55 or p75 TNFreceptor, sIL-1RI, sIL-1RII, sIL-6R), an antiinflammatory cytokine(e.g., IL-4, IL-10, IL-13 or TGFβ) or a bcl-2 inhibitor.

Examples of therapeutic agents for multiple sclerosis in which bindingproteins provided herein can be combined include interferon-13, forexample, IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspaseinhibitors, for example inhibitors of caspase-1, IL-1 inhibitors, TNFinhibitors, and antibodies to CD40 ligand and CD80.

Non-limiting examples of therapeutic agents for asthma with whichbinding proteins provided herein can be combined include the following:albuterol, salmeterol/fluticasone, montelukast sodium, fluticasonepropionate, budesonide, prednisone, salmeterol xinafoate, levalbuterolhcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate,triamcinolone acetonide, beclomethasone dipropionate, ipratropiumbromide, azithromycin, pirbuterol acetate, prednisolone, theophyllineanhydrous, methylprednisolone sodium succinate, clarithromycin,zafirlukast, formoterol fumarate, influenza virus vaccine,methylprednisolone, amoxicillin trihydrate, flunisolide, allergyinjection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine,chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which bindingproteins provided herein can be combined include the following:albuterol sulfate/ipratropium, ipratropium bromide,salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasonepropionate, prednisone, theophylline anhydrous, methylprednisolonesodium succinate, montelukast sodium, budesonide, formoterol fumarate,triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin,beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxonesodium, amoxicillin trihydrate, gatifloxacin, zafirlukast,amoxicillin/clavulanate, flunisolide/menthol,chlorpheniramine/hydrocodone, metaproterenol sulfate,methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir,pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.

Non-limiting examples of therapeutic agents for psoriasis with whichbinding proteins provided herein can be combined include the following:small molecule inhibitor of KDR, small molecule inhibitor of Tie-2,calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, pimecrolimus, coal tar, diflorasone diacetate, etanerceptfolate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,methylprednisolone acetate, prednisone, sunscreen, halcinonide,salicylic acid, anthralin, clocortolone pivalate, coal extract, coaltar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone,diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/nalact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen,salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,pimecrolimus, PUVA, UVB, sulfasalazine.

Examples of therapeutic agents for SLE (Lupus) in which binding proteinsprovided herein can be combined include the following: NSAIDS, forexample, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2inhibitors, for example, Celecoxib, rofecoxib, valdecoxib;anti-malarials, for example, hydroxychloroquine; Steroids, for example,prednisone, prednisolone, budenoside, dexamethasone; Cytotoxics, forexample, azathioprine, cyclophosphamide, mycophenolate mofetil,methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, forexample Cellcept. Binding proteins provided herein may also be combinedwith agents such as sulfasalazine, 5-aminosalicylic acid, olsalazine,Imuran and agents which interfere with synthesis, production or actionof proinflammatory cytokines such as IL-1, for example, caspaseinhibitors like IL-1β converting enzyme inhibitors and IL-1 ra. Bindingproteins provided herein may also be used with T cell signalinginhibitors, for example, tyrosine kinase inhibitors; or molecules thattarget T cell activation molecules, for example, CTLA-4-IgG or anti-B7family antibodies, anti-PD-1 family antibodies. Binding proteinsprovided herein, can be combined with IL-11 or anti-cytokine antibodies,for example, fonotolizumab (anti-IFNg antibody), or anti-receptorreceptor antibodies, for example, anti-IL-6 receptor antibody andantibodies to B-cell surface molecules. Antibodies provided herein orantigen binding portion thereof may also be used with LJP 394(abetimus), agents that deplete or inactivate B-cells, for example,Rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody), TNFantagonists, for example, anti-TNF antibodies, Adalimumab (PCTPublication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571, TNFR-Igconstructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG (LENERCEPT)) and bcl-2inhibitors, because bcl-2 overexpression in transgenic mice has beendemonstrated to cause a lupus like phenotype (see Marquina Thepharmaceutical compositions provided herein may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding protein provided herein. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding protein may bedetermined by a person skilled in the art and may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the binding protein to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the antibody, orantibody binding portion, are outweighed by the therapeuticallybeneficial effects. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

V. Diagnostics

The disclosure herein also provides diagnostic applications including,but not limited to, diagnostic assay methods, diagnostic kits containingone or more binding proteins, and adaptation of the methods and kits foruse in automated and/or semi-automated systems. The methods, kits, andadaptations provided may be employed in the detection, monitoring,and/or treatment of a disease or disorder in an individual. This isfurther elucidated below.

A. Method of Assay

The present disclosure also provides a method for determining thepresence, amount or concentration of an analyte, or fragment thereof, ina test sample using at least one binding protein as described herein.Any suitable assay as is known in the art can be used in the method.Examples include, but are not limited to, immunoassays and/or methodsemploying mass spectrometry.

Immunoassays provided by the present disclosure may include sandwichimmunoassays, radioimmunoassay (RIA), enzyme immunoassay (EIA),enzyme-linked immunosorbent assay (ELISA), competitive-inhibitionimmunoassays, fluorescence polarization immunoassay (FPIA), enzymemultiplied immunoassay technique (EMIT), bioluminescence resonanceenergy transfer (BRET), and homogenous chemiluminescent assays, amongothers.

A chemiluminescent microparticle immunoassay, in particular oneemploying the ARCHITECT® automated analyzer (Abbott Laboratories, AbbottPark, Ill.), is an example of an immunoassay.

Methods employing mass spectrometry are provided by the presentdisclosure and include, but are not limited to MALDI (matrix-assistedlaser desorption/ionization) or by SELDI (surface-enhanced laserdesorption/ionization).

Methods for collecting, handling, processing, and analyzing biologicaltest samples using immunoassays and mass spectrometry would bewell-known to one skilled in the art, are provided for in the practiceof the present disclosure (US 2009-0311253 A1).

B. Kit

A kit for assaying a test sample for the presence, amount orconcentration of an analyte, or fragment thereof, in a test sample isalso provided. The kit comprises at least one component for assaying thetest sample for the analyte, or fragment thereof, and instructions forassaying the test sample for the analyte, or fragment thereof. The atleast one component for assaying the test sample for the analyte, orfragment thereof, can include a composition comprising a bindingprotein, as disclosed herein, and/or an anti-analyte binding protein (ora fragment, a variant, or a fragment of a variant thereof), which isoptionally immobilized on a solid phase.

Optionally, the kit may comprise a calibrator or control, which maycomprise isolated or purified analyte. The kit can comprise at least onecomponent for assaying the test sample for an analyte by immunoassayand/or mass spectrometry. The kit components, including the analyte,binding protein, and/or anti-analyte binding protein, or fragmentsthereof, may be optionally labeled using any art-known detectable label.The materials and methods for the creation provided for in the practiceof the present disclosure would be known to one skilled in the art (US2009-0311253 A1).

C. Adaptation of Kit and Method

The kit (or components thereof), as well as the method of determiningthe presence, amount or concentration of an analyte in a test sample byan assay, such as an immunoassay as described herein, can be adapted foruse in a variety of automated and semi-automated systems (includingthose wherein the solid phase comprises a microparticle), as described,for example, in U.S. Pat. Nos. 5,089,424 and 5,006,309, and ascommercially marketed, for example, by Abbott Laboratories (Abbott Park,Ill.) as ARCHITECT®.

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, for example, U.S. Pat. No. 5,294,404,PRISM®, EIA (bead), and Quantum™ II, as well as other platforms.Additionally, the assays, kits and kit components can be employed inother formats, for example, on electrochemical or other hand-held orpoint-of-care assay systems. The present disclosure is, for example,applicable to the commercial Abbott Point of Care (i-STAT®, AbbottLaboratories) electrochemical immunoassay system that performs sandwichimmunoassays. Immunosensors and their methods of manufacture andoperation in single-use test devices are described, for example in, U.S.Pat. Nos. 5,063,081, 7,419,821, and 7,682,833; and US Publication Nos.20040018577, 20060160164 and US 20090311253.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinare obvious and may be made using suitable equivalents without departingfrom the scope of the embodiments disclosed herein. Having now describedcertain embodiments in detail, the same will be more clearly understoodby reference to the following examples, which are included for purposesof illustration only and are not intended to be limiting.

EXAMPLES Example 1 Generation of an Anti-IL-17 Humanized MonoclonalAntibody h10F7 Example 1.2 Affinity Maturation of the HumanizedAnti-Human IL-17 Antibody h10F7

A humanized anti-human IL-17 antibody was previously disclosed. Thisantibody was subsequently affinity matured to improve its overallaffinity to human, cynomolgus monkey, and mouse IL-17. Several librarieswere made according to specifications below:

H1+H2 Library:

-   -   Limited mutagenesis at 7 residues at 30, 31, 33, 35, 53, 56, 57,        and 58.    -   Toggle between human germline and h10F7 sequences at positions        27, 48, 51, 52, 54, 67, and 69.

H3 Library:

-   -   Limited mutagenesis at 95-100c and 102.    -   Toggle between human germline and h10F7 sequences at 93.

LC Library 1:

-   -   Limited mutagenesis at 30, 31, 32, 34, 50, 53, 89, 91, 92, 93,        and 96.    -   Toggle between human germline and h10F7 sequence at positions 4,        24, 27, 29, 33, 36, 43, 47, 52, and 55.

LC library 2: constructed with an additional residue at position G28 inCDR1 to increase identity to human antibodies.

-   -   Limited mutagenesis at 28, 30, 31, 32, 34, 50, 53, 89, 91, 92,        93, and 96.    -   Toggle between human germline and h10F7 sequence at positions        24, 27, 29, 33, 37, 44, 48, 52, and 55.    -   One framework germ-lining mutation in FR1 (position 4) to be        tested first as scFv. “M” will be preferred over “L” if binding        is not affected.

rHC library: recombine outputs of H1+H2 and H3 libraries.

rHCLC library: recombine outputs of H1+H2, H3, and LC1 or LC2 libraries.(Choose LC2 over LC1 if LC2 output binding appears to be at least asgood as WT, otherwise recombine LC1 output).

All four libraries were selected separately for the ability to bindhuman, cynomolgus monkey and mouse IL-17 in the presence of decreasingconcentrations of biotinylated antigens. All mutated CDR sequencesrecovered from library selections were recombined into additionallibraries and the recombined libraries were subjected to more stringentselection conditions before individual antibodies are identified.

The table below provides a list of amino acid sequences of VH and VL ofthe h10F7 antibody which were subjected to the affinity maturationselection protocol. Amino acid residues of individual CDRs of each VHand VL sequence are indicated in bold.

TABLE 2  Amino acid residues observed in affinitymatured anti-IL-17 antibody h10F7 h10f7 Heavy chain variable regionh10f7VH.1 1234567890123456789012345678901234567890123456789012a345678901EVQLVQSGAEVKKPGSSVKVSCKASGYTFT DYEIH WVRQAPGQGLEWIG VNDPESGGIFYNQ               DE        M I D  SL                S              IY               A              M                E                 N                P 234567890123456789012abc345678901234567890abcd1234567890123 KFDGRATLTADKSTSTAYMELSSLRSEDTAVYYCTR YYRYESFYGMDY WGQGTTVIVSS   V I               DKWDGLE  I    I                SMFW YN  C                   W Y SD  E                    F V L  L                   E N S  S                    N M Q  F                    F A H                       R A                      F                       M                       K                      H h10f7 Light chain variable region H10f7Vk.1a1234567890123456789012345678901234567890123456789012345678901DIQLTQSPSSLSASVGDRVTITC SASS-SISYIY WFQQKPGKSPKRWIY ATFELAS GVPSR  M          R QGIRRCLD Y   A  L R SD Q            K  SVSNSVN        E A               DLVGD F        D Y              Q NTN S        S G               A FPP Q        G Q              I GLI G        T W               V LDF         Q L              T PCA         P H               E K          M               M 234567890123456789012345678901234567890123456aFSGSGSGTDYTLTISSLQPEDFATYYC HQRSSYPWT FGQGTKLEIK               R VGN E              Q WTI L               L LMR G               N MNF F              Y KRT Y               S FKY P               V PAL I              A GDM D                S W R                Q K H               I H Q                T G N                D  C

Tables 1B and 1C in section I.C above, provide individual VH and VLsequences from converted clones. These affinity matured sequences wereconverted to full length IgG.

TABLE 3 h10F7 affinity matured scFv clones converted to full length IgGFull length IgG ScFv clone name HC plasmid LC plasmid (protein) nameJ427 M2S3 #12 pHybE- pHybE-hCk V3 h10F7-M12 h10F7VH.1a.g1m J427 M2S3 #12(SEQ ID NO: 42) (SEQ ID NO: 52) J427 M2S3 #27 pHybE- pHybE-hCk V3h10F7-M27 h10F7VH.1a.g1m J427 M2S3 #27 (SEQ ID NO: 42) (SEQ ID NO: 53)J439M1S3R5#10 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-M10 a, mut (234,235) V2 J439M1S3R5#10 J439M1S3R5#10 (SEQ ID NO: 50) (SEQ ID NO: 43)J439M1S3R5#11 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-M11 a, mut (234,235) V2 J439M1S3R5#11 J439M1S3R5#11 (SEQ ID NO: 51) (SEQ ID NO: 44)J439M1S2(H)3 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-A6 #A6 a, mut (234,235) V2 J439M1S2(H)3#A6 J439M1S2(H)3 #A6 (SEQ ID NO: 54) (SEQ ID NO: 45)J439M1S2(H)3 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-A11 #A11 a, mut(234, 235) V2 J439M1S3R5#11 J439M1S2(H)3 #A11 (SEQ ID NO: 51) (SEQ IDNO: 46) J439M1S2(H)3 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-A16 #A16 a,mut (234, 235) V2 J439M1S2(H)3#A6 J439M1S2(H)3 #A16 (SEQ ID NO: 54) (SEQID NO: 47) J439M1S2(H)3 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-B13 #B13a, mut (234, 235) V2 J439M1S3R5#11 J439M1S2(H)3 #B13 (SEQ ID NO: 51)(SEQ ID NO: 48) J439M1S2(H)3 pHybE-hCg1, z, non- pHybE-hCk V3 h10F7-B20#B20 a, mut (234, 235) V2 J439M1S3R5#11 J439M1S2(H)3 #B20 (SEQ ID NO:51) (SEQ ID NO: 49)

Example 2 Generation and Characterization of Anti-IL-13 and Anti-IL-17Dual Variable Domain (DVD) Binding Proteins

Four-chain dual variable domain (DVD) binding proteins using parentantibodies with known amino acid sequences were generated bysynthesizing polynucleotide fragments encoding DVD binding proteinvariable heavy and DVD binding protein variable light chain sequencesand cloning the fragments into a pHybC-D2 vector according to art knownmethods. The DVD binding protein constructs were cloned into andexpressed in 293 cells and purified according to art known methods. DVDVH and VL chains for the DVD binding proteins are provided below.

Example 2.1 DVD Binding Proteins that Bind IL-13 and IL-17

TABLE 4 DVD Outer Variable Variable Inner Domain Domain Variable SEQ IDVD1 - X1 - Name Name Linker Domain VD2 Formula DVD2148H AB397VH HG-shortAB273VH 30 - 21 - 36 DVD2148L AB397VL LK-short AB273VL 31 - 13 - 37DVD2149H AB273H HG-short AB397VH 36 - 21 - 30 DVD2149L AB273L LK-shortAB397VL 37 - 13 - 31 DVD2150H AB397VH HG-short AB273VH 30 - 21 - 36DVD2150L AB397VL LK-long AB273VL 31 - 14 - 37 DVD2151H AB273VH HG-shortAB397VH 36 - 21 - 30 DVD2151L AB273VL LK-long AB397VL 37 - 14 - 31DVD2152H AB397VH HG-long AB273VH 30 - 22 - 36 DVD2152L AB397VL LK-shortAB273VL 31 - 13 - 37 DVD2153H AB273VH HG-long AB397VH 36 - 22 - 30DVD2153L AB273VL LK-short AB397VL 37 - 13 - 31 DVD2154H AB397VH HG-shortAB274VH 30 - 21 - 38 DVD2154L AB397VL LK-short AB274VL 31 - 13 - 39DVD2155H AB274VH HG-short AB397VH 38 - 21 - 30 DVD2155L AB274VL LK-shortAB397VL 39 - 13 - 31 DVD2156H AB397VH HG-short AB274VH 30 - 21 - 38DVD2156L AB397VL LK-long AB274VL 31 - 14 - 39 DVD2157H AB274VH HG-shortAB397VH 38 - 21 - 30 DVD2157L AB274VL LK-long AB397VL 39 - 14 - 31DVD2158H AB397VH HG-long AB274VH 30 - 22 - 38 DVD2158L AB397VL LK-shortAB274VL 31 - 13 - 39 DVD2159H AB274VH HG-long AB397VH 38 - 22 - 30DVD2159L AB274VL LK-short AB397VL 39 - 13 - 31 DVD2160H AB397VH HG-shortAB275VH 30 - 21 - 40 DVD2160L AB397VL LK-short AB275VL 31 - 13 - 40DVD2161H AB275VH HG-short AB397VH 40 - 21 - 30 DVD2161L AB275VL LK-shortAB397VL 41 - 13 - 31 DVD2162H AB397VH HG-short AB275VH 30 - 21 - 40DVD2162L AB397VL LK-long AB275VL 31 - 14 - 41 DVD2163H AB275VH HG-shortAB397VH 40 - 21 - 30 DVD2163L AB275VL LK-long AB397VL 41 - 14 - 31DVD2164H AB397VH HG-long AB275VH 30 - 22 - 40 DVD2164L AB397VL LK-shortAB275VL 31 - 13 - 41 DVD2165H AB275VH HG-long AB397VH 40 - 22 - 30DVD2165L AB275VL LK-short AB397VL 41 - 13 - 31 DVD2166H AB398VH HG-shortAB273VH 32 - 21 - 36 DVD2166L AB398VL LK-short AB273VL 33 - 13 - 37DVD2167H AB273VH HG-short AB398VH 36 - 21 - 32 DVD2167L AB273VL LK-shortAB398VL 37 - 13 - 33 DVD2168H AB398VH HG-short AB273VH 32 - 21 - 36DVD2168L AB398VL LK-long AB273VL 33 - 14 - 37 DVD2169H AB273VH HG-shortAB398VH 36 - 21 - 32 DVD2169L AB273VL LK-long AB398VL 37 - 14 - 33DVD2170H AB398VH HG-long AB273VH 32 - 22 - 36 DVD2170L AB398VL LK-shortAB273VL 33 - 13 - 37 DVD2171H AB273VH HG-long AB398VH 36 - 22 - 32DVD2171L AB273VL LK-short AB398VL 37 - 13 - 33 DVD2172H AB398VH HG-shortAB274VH 32 - 21 - 38 DVD2172L AB398VL LK-short AB274VL 33 - 13 - 39DVD2173H AB274VH HG-short AB398VH 38 - 21 - 32 DVD2173L AB274VL LK-shortAB398VL 39 - 13 - 33 DVD2174H AB398VH HG-short AB274VH 32 - 21 - 38DVD2174L AB398VL LK-long AB274VL 33 - 14 - 39 DVD2175H AB274VH HG-shortAB398VH 38 - 21 - 32 DVD2175L AB274VL LK-long AB398VL 39 - 14 - 33DVD2176H AB398VH HG-long AB274VH 32 - 22 - 38 DVD2176L AB398VL LK-shortAB274VL 33 - 13 - 39 DVD2177H AB274VH HG-long AB398VH 38 - 22 - 32DVD2177L AB274VL LK-short AB398VL 39 - 13 - 33 DVD2178H AB398VH HG-shortAB275VH 32 - 21 - 40 DVD2178L AB398VL LK-short AB275VL 33 - 13 - 41DVD2179H AB275VH HG-short AB398VH 40 - 21 - 32 DVD2179L AB275VL LK-shortAB398VL 41 - 13 - 33 DVD2180H AB398VH HG-short AB275VH 32 - 21 - 40DVD2180L AB398VL LK-long AB275VL 33 - 14 - 41 DVD2181H AB275VH HG-shortAB398VH 40 - 21 - 32 DVD2181L AB275VL LK-long AB398VL 41 - 14 - 43DVD2182H AB398VH HG-long AB275VH 32 - 22 - 40 DVD2182L AB398VL LK-shortAB275VL 33 - 13 - 41 DVD2183H AB275VH HG-long AB398VH 40 - 22 - 32DVD2183L AB275VL LK-short AB398VL 41 - 13 - 33 DVD2184H AB399VH HG-shortAB273VH 34 - 21 - 36 DVD2184L AB399VL LK-short AB273VL 35 - 13 - 37DVD2185H AB273VH HG-short AB399VH 36 - 21 - 34 DVD2185L AB273VL LK-shortAB399VL 37 - 13 - 35 DVD2186H AB399VH HG-short AB273VH 34 - 21 - 36DVD2186L AB399VL LK-long AB273VL 35 - 14 - 37 DVD2187H AB273VH HG-shortAB399VH 36 - 21 - 34 DVD2187L AB273VL LK-long AB399VL 37 - 14 - 35DVD2188H AB399VH HG-long AB273VH 34 - 22 - 36 DVD2188L AB399VL LK-shortAB273VL 35 - 13 - 37 DVD2189H AB273VH HG-long AB399VH 36 - 22 - 34DVD2189L AB273VL LK-short AB399VL 37 - 13 - 35 DVD2190H AB399VH HG-shortAB274VH 34 - 21 - 38 DVD2190L AB399VL LK-short AB274VL 35 - 13 - 39DVD2191H AB274VH HG-short AB399VH  8 - 21 - 34 DVD2191L AB274VL LK-shortAB399VL 39 - 13 - 35 DVD2192H AB399VH HG-short AB274VH 34 - 21 - 38DVD2192L AB399VL LK-long AB274VL 35 - 14 - 39 DVD2193H AB274VH HG-shortAB399VH 38 - 21 - 34 DVD2193L AB274VL LK-long AB399VL 39 - 14 - 35DVD2194H AB399VH HG-long AB274VH 34 - 22 - 38 DVD2194L AB399VL LK-shortAB274VL 35 - 13 - 39 DVD2195H AB274VH HG-long AB399VH 38 - 22 - 34DVD2195L AB274VL LK-short AB399VL 39 - 13 - 35 DVD2196H AB399VH HG-shortAB275VH 34 - 21 - 40 DVD2196L AB399VL LK-short AB275VL 35 - 13 - 41DVD2197H AB275VH HG-short AB399VH 40 - 21 - 34 DVD2197L AB275VL LK-shortAB399VL 41 - 13 - 35 DVD2198H AB399VH HG-short AB275VH 34 - 21 - 40DVD2198L AB399VL LK-long AB275VL 35 - 14 - 41 DVD2199H AB275VH HG-shortAB399VH 40 - 21 - 34 DVD2199L AB275VL LK-long AB399VL 41 - 14 - 35DVD2200H AB399VH HG-long AB275VH 34 - 22 - 40 DVD2200L AB399VL LK-shortAB275VL 35 - 13 - 41 DVD2201H AB275VH HG-long AB399VH 40 - 22 - 34DVD2201L AB275VL LK-short AB399VL 41 - 13 - 35

Table 5 contains the yield data for parent antibodies and DVD-Igconstructs expressed as milligrams per liter in 293 cells.

TABLE 5 Transient Expression in Yields of Parent Antibodies and DVD-IgConstructs in 293 Cells Parent N-terminal C-terminal Antibody orVariable Variable Expression DVD-Ig ID Domain (VD) Domain (VD) yield(mg/L) AB273 IL-17 (SEQ 1) 16.6 AB274 IL-17 (SEQ 2) 44.6 AB275 IL-17(SEQ 3) 44.2 AB397 IL-13 (SEQ 1) 73.6 AB398 IL-13 (SEQ 2) 96.0 AB399IL-13 (SEQ 3) 83.8 DVD2148 IL-13 (SEQ 1) IL-17 (SEQ 1) 14.4 DVD2149IL-17 (SEQ 1) IL-13 (SEQ 1) 0.6 DVD2150 IL-13 (SEQ 1) IL-17 (SEQ 1) 6.2DVD2151 IL-17 (SEQ 1) IL-13 (SEQ 1) 0.4 DVD2152 IL-13 (SEQ 1) IL-17(SEQ 1) 25.6 DVD2153 IL-17 (SEQ 1) IL-13 (SEQ 1) 1.0 DVD2154 IL-13(SEQ 1) IL-17 (SEQ 2) 35.0 DVD2155 IL-17 (SEQ 2) IL-13 (SEQ 1) 30.8DVD2156 IL-13 (SEQ 1) IL-17 (SEQ 2) 37.2 DVD2157 IL-17 (SEQ 2) IL-13(SEQ 1) 29.2 DVD2158 IL-13 (SEQ 1) IL-17 (SEQ 2) 57.2 DVD2159 IL-17 (SEQ2) IL-13 (SEQ 1) 37.4 DVD2160 IL-13 (SEQ 1) IL-17 (SEQ 3) 65.8 DVD2161IL-17 (SEQ 3) IL-13 (SEQ 1) 47.2 DVD2162 IL-13 (SEQ 1) IL-17 (SEQ 3)38.2 DVD2163 IL-17 (SEQ 3) IL-13 (SEQ 1) 33.6 DVD2164 IL-13 (SEQ 1)IL-17 (SEQ 3) 64.4 DVD2165 IL-17 (SEQ 3) IL-13 (SEQ 1) 48.5 DVD2166IL-13 (SEQ 2) IL-17 (SEQ 1) 13.7 DVD2167 IL-17 (SEQ 1) IL-13 (SEQ 2) 8.9DVD2168 IL-13 (SEQ 2) IL-17 (SEQ 1) 7.8 DVD2169 IL-17 (SEQ 1) IL-13 (SEQ2) 1.9 DVD2170 IL-13 (SEQ 2) IL-17 (SEQ 1) 10.4 DVD2171 IL-17 (SEQ 1)IL-13 (SEQ 2) 4.3 DVD2172 IL-13 (SEQ 2) IL-17 (SEQ 2) 26.5 DVD2173 IL-17(SEQ 2) IL-13 (SEQ 2) 16.5 DVD2174 IL-13 (SEQ 2) IL-17 (SEQ 2) 28.3DVD2175 IL-17 (SEQ 2) IL-13 (SEQ 2) 10.7 DVD2176 IL-13 (SEQ 2) IL-17(SEQ 2) 21.5 DVD2177 IL-17 (SEQ 2) IL-13 (SEQ 2) 18.4 DVD2178 IL-13 (SEQ2) IL-17 (SEQ 3) 9.5 DVD2179 IL-17 (SEQ 3) IL-13 (SEQ 2) 8.9 DVD2180IL-13 (SEQ 2) IL-17 (SEQ 3) 8.5 DVD2181 IL-17 (SEQ 3) IL-13 (SEQ 2) 2.8DVD2182 IL-13 (SEQ 2) IL-17 (SEQ 3) 14.0 DVD2183 IL-17 (SEQ 3) IL-13(SEQ 2) 9.2 DVD2184 IL-13 (SEQ 3) IL-17 (SEQ 1) 0.2 DVD2185 IL-17(SEQ 1) IL-13 (SEQ 3) 26.9 DVD2186 IL-13 (SEQ 3) IL-17 (SEQ 1) 0.0DVD2187 IL-17 (SEQ 1) IL-13 (SEQ 3) 2.3 DVD2188 IL-13 (SEQ 3) IL-17(SEQ 1) 0.0 DVD2189 IL-17 (SEQ 1) IL-13 (SEQ 3) 1.3 DVD2190 IL-13 (SEQ3) IL-17 (SEQ 2) 31.5 DVD2191 IL-17 (SEQ 2) IL-13 (SEQ 3) 42.7 DVD2192IL-13 (SEQ 3) IL-17 (SEQ 2) 2.6 DVD2193 IL-17 (SEQ 2) IL-13 (SEQ 3) 18.3DVD2194 IL-13 (SEQ 3) IL-17 (SEQ 2) 0.0 DVD2195 IL-17 (SEQ 2) IL-13 (SEQ3) 21.6 DVD2196 IL-13 (SEQ 3) IL-17 (SEQ 3) 27.4 DVD2197 IL-17 (SEQ 3)IL-13 (SEQ 3) 72.2 DVD2198 IL-13 (SEQ 3) IL-17 (SEQ 3) 0.1 DVD2199 IL-17(SEQ 3) IL-13 (SEQ 3) 8.1 DVD2200 IL-13 (SEQ 3) IL-17 (SEQ 3) 1.1DVD2201 IL-17 (SEQ 3) IL-13 (SEQ 3) 11.4 All DVD-Ig molecules expressedwell in 293 cells. DVD-Ig molecules could be easily purified over aprotein A column. In most cases, >5 mg/L purified DVD-Ig protein couldbe obtained easily from supernatants of 293 cells.

Example 3 Assays Used to Determine the Functional Activity of ParentAntibodies and DVD-Ig Proteins Example 3.1 IL-13 Bioassay andNeutralization Assay

A549 cells were plated at 1.5−2×10⁵ cells per well in a 100 μL volumeand incubated overnight at 37° C., 5% CO₂. Following a 16-20 hourovernight incubation, the original 100 μl media seeding volume wasremoved and 100 μL of 400 ng/mL (2× concentrated) rhTNF-α was added toall wells. The plates were placed at 37° C., 5% CO₂ until the additionof IL-13 and antibody or DVD-Ig protein. A 20 μg/mL working stock ofantibody or DVD-Ig protein (4× concentrated) was prepared in completeF12 medium. An eight point serial dilution was performed (5 μg/mL-0.0003μg/mL) in complete F12 in Marsh dilution plates. Sixty uL/well of eachantibody or DVD-Ig protein dilution was added in quadruplicate to a 96well v-bottom (Costar#3894) plate and 60 μL of a 4× concentrated (20ng/mL) solution of IL-13 was added to all wells except the cell onlycontrol. Following a 1 hour incubation, 100 μL of the aboveIL-13/Antibody or DVD-Ig protein complex was added to the A549 cells.All well volumes were equal to 200 μL. The final concentration ofrecombinant IL-13 was 5 ng/mL and rhTNF-α was 200 ng/mL. All platereagents were then 1× concentrated. After a 16-20 hour incubation, thewell contents (200 μL) were transferred into a 96-well round bottomplate (Costar#3799) and placed in a −20° C. freezer. The supernatantswere tested for hTARC levels by ELISA in the Assay Lab. Neutralizationpotency was determined by calculating percent inhibition relative to the5 ng/mL IL-13 alone control value. Reported IC₅₀ values (sigmoidal curvedose responses) were calculated using GraphPad Prism.

TABLE 6 IL-13 Neutralization Assay With IL-13 Parent Antibody and DVD-IgProtein N-Terminal C-Terminal N-terminal C-terminal VD IL13 VD IL13Parent Antibody Variable Variable Neutralization Neutralization orDVD-Ig ID Domain (VD) Domain (VD) Assay IC50 nM AssayIC50 nM AB397 IL-13(SEQ 1) 0.033 AB398 IL-13 (SEQ 2) 0.082 AB399 IL-13 (SEQ 3) 0.043DVD2148 IL-13 (SEQ 1) IL-17 (SEQ 1) 0.054 NA DVD2149 IL-17 (SEQ 1) IL-13(SEQ 1) NA 1.426 DVD2150 IL-13 (SEQ 1) IL-17 (SEQ 1) 0.099 NA DVD2151IL-17 (SEQ 1) IL-13 (SEQ 1) NA 0.526 DVD2152 IL-13 (SEQ 1) IL-17 (SEQ 1)0.112 NA DVD2153 IL-17 (SEQ 1) IL-13 (SEQ 1) NA 2.442 DVD2154 IL-13(SEQ 1) IL-17 (SEQ 2) 0.430 NA DVD2155 IL-17 (SEQ 2) IL-13 (SEQ 1) NA1.180 DVD2156 IL-13 (SEQ 1) IL-17 (SEQ 2) 0.129 NA DVD2157 IL-17 (SEQ 2)IL-13 (SEQ 1) NA 0.244 DVD2158 IL-13 (SEQ 1) IL-17 (SEQ 2) 0.131 NADVD2159 IL-17 (SEQ 2) IL-13 (SEQ 1) NA 0.876 DVD2160 IL-13 (SEQ 1) IL-17(SEQ 3) 0.178 NA DVD2161 IL-17 (SEQ 3) IL-13 (SEQ 1) NA 0.812 DVD2162IL-13 (SEQ 1) IL-17 (SEQ 3) 0.126 NA DVD2163 IL-17 (SEQ 3) IL-13 (SEQ 1)NA 0.195 DVD2164 IL-13 (SEQ 1) IL-17 (SEQ 3) 0.146 NA DVD2165 IL-17 (SEQ3) IL-13 (SEQ 1) NA 0.355 DVD2166 IL-13 (SEQ 2) IL-17 (SEQ 1) 0.089 NADVD2167 IL-17 (SEQ 1) IL-13 (SEQ 2) NA 1.700 DVD2168 IL-13 (SEQ 2) IL-17(SEQ 1) 0.119 NA DVD2169 IL-17 (SEQ 1) IL-13 (SEQ 2) NA 0.309 DVD2170IL-13 (SEQ 2) IL-17 (SEQ 1) 0.106 NA DVD2171 IL-17 (SEQ 1) IL-13 (SEQ 2)NA 0.506 DVD2172 IL-13 (SEQ 2) IL-17 (SEQ 2) 0.111 NA DVD2173 IL-17 (SEQ2) IL-13 (SEQ 2) NA 2.136 DVD2174 IL-13 (SEQ 2) IL-17 (SEQ 2) 0.085 NADVD2175 IL-17 (SEQ 2) IL-13 (SEQ 2) NA 0.115 DVD2176 IL-13 (SEQ 2) IL-17(SEQ 2) 0.106 NA DVD2177 IL-17 (SEQ 2) IL-13 (SEQ 2) NA 0.155 DVD2178IL-13 (SEQ 2) IL-17 (SEQ 3) 0.088 NA DVD2179 IL-17 (SEQ 3) IL-13 (SEQ 2)NA 0.916 DVD2180 IL-13 (SEQ 2) IL-17 (SEQ 3) 0.091 NA DVD2181 IL-17 (SEQ3) IL-13 (SEQ 2) NA 0.040 DVD2182 IL-13 (SEQ 2) IL-17 (SEQ 3) 0.077 NADVD2183 IL-17 (SEQ 3) IL-13 (SEQ 2) NA 0.086 DVD2184 IL-13 (SEQ 3) IL-17(SEQ 1) 0.121 NA DVD2185 IL-17 (SEQ 1) IL-13 (SEQ 3) NA 1.254 DVD2187IL-17 (SEQ 1) IL-13 (SEQ 3) NA 0.811 DVD2189 IL-17 (SEQ 1) IL-13 (SEQ 3)NA 0.172 DVD2190 IL-13 (SEQ 3) IL-17 (SEQ 2) 0.087 NA DVD2191 IL-17 (SEQ2) IL-13 (SEQ 3) NA 0.165 DVD2192 IL-13 (SEQ 3) IL-17 (SEQ 2) 0.056 NADVD2193 IL-17 (SEQ 2) IL-13 (SEQ 3) NA 0.028 DVD2195 IL-17 (SEQ 2) IL-13(SEQ 3) NA 0.052 DVD2196 IL-13 (SEQ 3) IL-17 (SEQ 3) 0.029 NA DVD2197IL-17 (SEQ 3) IL-13 (SEQ 3) NA 0.081 DVD2199 IL-17 (SEQ 3) IL-13 (SEQ 3)NA 0.088 DVD2200 IL-13 (SEQ 3) IL-17 (SEQ 3) 0.096 NA DVD2201 IL-17 (SEQ3) IL-13 (SEQ 3) NA 0.092 All DVD-Ig proteins containing VDs from AB397,AB398, or AB399 in either the N-terminal or C-terminal position showedneutralization in the A549 IL-13 neutralization assay.

Example 3.2 IL-17 Bioassay and Neutralization Assay

The human HS27 cell line (ATCC #CRL-1634) secretes IL-6 in response toIL-17. The IL-17-induced IL-6 secretion is inhibited by neutralizinganti-IL-17 antibodies (See, e.g., J. Imm. 155:5483-5486 (1995) orCytokine 9:794-800 (1997)).

HS27 cells were maintained in assay medium (DMEM high glucose medium(Gibco #11965) with 10% fetal bovine serum (Gibco#26140), 4 mML-glutamine, 1 mM sodium pyruvate, penicillin G (100 U/500 ml) andstreptomycin (100 μg/500 ml)). Cells were grown in T150 flasks untilthey were about 80-90% confluent the day of the assay. Human IL-17 (R&DSystems, #317-IL/CF) was reconstituted in sterile PBS without Ca²⁺ andMg²⁺, stored frozen, freshly thawed for use and diluted to 40 ng/ml (4×)in assay medium. Serial dilutions of antibodies were made in a separateplate (4× concentrations), mixed with an equal volume of 40 ng/ml (4×)of human IL-17 and incubated at 37° C. for 1 hour. HS27 cells (typicallyabout 20,000 cells in 50 μl assay medium) were added to each well of a96-well flat-bottom tissue culture plate (Costar #3599), followed by theaddition of 50 μl of the pre-incubated antibody or DVD-Ig protein plusIL-17 mix. The final concentration of IL-17 was 10 ng/ml. Cells wereincubated for about 24 hours at 37° C. The media supernatants were thencollected. The level of IL-17 neutralization was measured bydetermination of IL-6 amounts in supernatant using a commercial MesoScale Discovery kit according to manufacturer's instructions. IC50values were obtained using logarithm of antibody or DVD-Ig protein vs.IL-6 amount variable slope fit.

TABLE 7 IL-17 Neutralization Assay With IL-17 Parent Antibody and DVD-IgProtein N-Terminal C-Terminal N-terminal C-terminal VD IL17 VD IL17Parent Antibody Variable Variable Neutralization Neutralization orDVD-Ig ID Domain (VD) Domain (VD) Assay IC50 nM Assay IC50 nM AB273IL-17 (SEQ 1) 0.0198 AB274 IL-17 (SEQ 2) 0.003 AB275 IL-17 (SEQ 3)0.0052 DVD2148 IL-13 (SEQ 1) IL-17 (SEQ 1) NA 0.0116 DVD2149 IL-17(SEQ 1) IL-13 (SEQ 1) 0.0028 NA DVD2150 IL-13 (SEQ 1) IL-17 (SEQ 1) NA0.004  DVD2151 IL-17 (SEQ 1) IL-13 (SEQ 1) 0.0179 NA DVD2152 IL-13(SEQ 1) IL-17 (SEQ 1) NA 0.0069 DVD2153 IL-17 (SEQ 1) IL-13 (SEQ 1)0.0049 NA DVD2154 IL-13 (SEQ 1) IL-17 (SEQ 2) NA 0.3508 DVD2155 IL-17(SEQ 2) IL-13 (SEQ 1) 0.0078 NA DVD2156 IL-13 (SEQ 1) IL-17 (SEQ 2) NA0.0155 DVD2157 IL-17 (SEQ 2) IL-13 (SEQ 1) 0.0066 NA DVD2158 IL-13(SEQ 1) IL-17 (SEQ 2) NA 0.0998 DVD2159 IL-17 (SEQ 2) IL-13 (SEQ 1)0.0081 NA DVD2160 IL-13 (SEQ 1) IL-17 (SEQ 3) NA 0.7034 DVD2161 IL-17(SEQ 3) IL-13 (SEQ 1) 0.0144 NA DVD2162 IL-13 (SEQ 1) IL-17 (SEQ 3) NA0.0213 DVD2163 IL-17 (SEQ 3) IL-13 (SEQ 1) 0.0151 NA DVD2164 IL-13(SEQ 1) IL-17 (SEQ 3) NA 0.1354 DVD2165 IL-17 (SEQ 3) IL-13 (SEQ 1)0.0133 NA DVD2166 IL-13 (SEQ 2) IL-17 (SEQ 1) NA 0.0164 DVD2167 IL-17(SEQ 1) IL-13 (SEQ 2) 0.0029 NA DVD2168 IL-13 (SEQ 2) IL-17 (SEQ 1) NA0.0025 DVD2169 IL-17 (SEQ 1) IL-13 (SEQ 2) 0.0021 NA DVD2170 IL-13 (SEQ2) IL-17 (SEQ 1) NA 0.0053 DVD2171 IL-17 (SEQ 1) IL-13 (SEQ 2) 0.0011 NADVD2172 IL-13 (SEQ 2) IL-17 (SEQ 2) NA 0.6781 DVD2173 IL-17 (SEQ 2)IL-13 (SEQ 2) 0.008  NA DVD2174 IL-13 (SEQ 2) IL-17 (SEQ 2) NA 0.0126DVD2175 IL-17 (SEQ 2) IL-13 (SEQ 2) 0.0089 NA DVD2176 IL-13 (SEQ 2)IL-17 (SEQ 2) NA 0.1164 DVD2177 IL-17 (SEQ 2) IL-13 (SEQ 2) 0.0101 NADVD2178 IL-13 (SEQ 2) IL-17 (SEQ 3) NA 0.6911 DVD2179 IL-17 (SEQ 3)IL-13 (SEQ 2) 0.0097 NA DVD2180 IL-13 (SEQ 2) IL-17 (SEQ 3) NA 0.0567DVD2181 IL-17 (SEQ 3) IL-13 (SEQ 2) 0.0097 NA DVD2182 IL-13 (SEQ 2)IL-17 (SEQ 3) NA 0.266  DVD2183 IL-17 (SEQ 3) IL-13 (SEQ 2) 0.0125 NADVD2184 IL-13 (SEQ 3) IL-17 (SEQ 1) NA 0.4054 DVD2185 IL-17 (SEQ 1)IL-13 (SEQ 3) 0.0013 NA DVD2187 IL-17 (SEQ 1) IL-13 (SEQ 3) 0.0016 NADVD2189 IL-17 (SEQ 1) IL-13 (SEQ 3) 0.0017 NA DVD2190 IL-13 (SEQ 3)IL-17 (SEQ 2) NA 0.061  DVD2191 IL-17 (SEQ 2) IL-13 (SEQ 3) 0.013  NADVD2192 IL-13 (SEQ 3) IL-17 (SEQ 2) NA 0.015  DVD2193 IL-17 (SEQ 2)IL-13 (SEQ 3) 0.014  NA DVD2195 IL-17 (SEQ 2) IL-13 (SEQ 3) 0.0181 NADVD2196 IL-13 (SEQ 3) IL-17 (SEQ 3) NA 0.0551 DVD2197 IL-17 (SEQ 3)IL-13 (SEQ 3) 0.005  NA DVD2199 IL-17 (SEQ 3) IL-13 (SEQ 3) 0.0102 NADVD2200 IL-13 (SEQ 3) IL-17 (SEQ 3) NA 0.0072 DVD2201 IL-17 (SEQ 3)IL-13 (SEQ 3) 0.0116 NA All DVD-Ig proteins containing VDs from AB273,AB274, or AB275 in either the N-terminal or C-terminal position showedneutralization in the HS27 IL-17 neutralization assay.

Example 3.3 Affinity Determination Using BIACORE Technology

TABLE 8 Reagents Used in Biacore Analyses Assay Antigen VendorDesignation Vendor Catalog # IL-17 Recombinant Human IL-17 R&D 317-ILsystems IL-13 Recombinant Human IL-13 R&D 213-IL systems

BIACORE Methods:

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determined theaffinity of antibodies or DVD-Ig with kinetic measurements of on-rateand off-rate constants. Binding of antibodies or DVD-Ig proteins to atarget antigen (for example, a purified recombinant target antigen) wasdetermined by surface plasmon resonance-based measurements with aBiacore®1000 or 3000 instrument (Biacore® AB, Uppsala, Sweden) usingrunning HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005%surfactant P20) at 25° C. All chemicals were obtained from Biacore® AB(Uppsala, Sweden) or otherwise from a different source as described inthe text. For example, approximately 5000 RU of goat anti-mouse IgG,(Fcγ), fragment specific polyclonal antibody (Pierce Biotechnology Inc,Rockford, Ill.) diluted in 10 mM sodium acetate (pH 4.5) was directlyimmobilized across a CM5 research grade biosensor chip using a standardamine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface wereblocked with ethanolamine. Modified carboxymethyl dextran surface inflowcell 2 and 4 was used as a reaction surface. Unmodifiedcarboxymethyl dextran without goat anti-mouse IgG in flow cell 1 and 3was used as the reference surface. For kinetic analysis, rate equationsderived from the 1:1 Langmuir binding model were fitted simultaneouslyto association and dissociation phases of all eight injections (usingglobal fit analysis) with the use of Biaevaluation 4.0.1 software.Purified antibodies or DVD-Ig proteins were diluted in HEPES-bufferedsaline for capture across goat anti-mouse IgG specific reactionsurfaces. Antibodies or DVD-Ig proteins to be captured as a ligand (25μg/ml) were injected over reaction matrices at a flow rate of 5μl/minute. The association and dissociation rate constants, k_(on)(M⁻¹s⁻¹) and k_(off) (s⁻¹) were determined under a continuous flow rateof 25 μl/minute. Rate constants were derived by making kinetic bindingmeasurements at different antigen concentrations ranging from 10-200 nM.The equilibrium dissociation constant (M) of the reaction betweenantibodies or DVD-Ig proteins and the target antigen was then calculatedfrom the kinetic rate constants by the following formula:K_(D)=k_(off)/k_(on). Binding was recorded as a function of time andkinetic rate constants were calculated. In this assay, on-rates as fastas 10⁶M⁻¹s⁻¹ and off-rates as slow as 10⁻⁶ s⁻¹ were measured.

TABLE 9 BIACORE Analysis of Parental Antibodies and DVD-Ig ProteinsN-terminal C-terminal Parent Antibody Variable Variable k_(on) k_(off)k_(D) or DVD-Ig ID Domain (VD) Domain (VD) (M−1s−1) (s−1) (M) AB273IL-17 (SEQ 1) 2.00E+06 <1.0e−6     <5.0E−13  AB274 IL-17 (SEQ 2)1.10E+06 8.20E−06 7.40E−12 AB275 IL-17 (SEQ 3) 1.90E+06 6.50E−053.40E−11 AB397 IL-13 (SEQ 1) 9.20E+05 1.20E−04 1.30E−10 AB398 IL-13 (SEQ2) 6.30E+05 2.80E−05 4.50E−11 AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−051.90E−11 AB397 IL-13 (SEQ 1) 9.20E+05 1.20E−04 1.30E−10 AB274 IL-17 (SEQ2) 1.10E+06 8.20E−06 7.40E−12 DVD2155 IL-17 (SEQ 2) 2.70E+06 6.90E−062.60E−12 DVD2155 IL-13 (SEQ 1) 2.30E+05 3.80E−05 1.60E−10 AB397 IL-13(SEQ 1) 9.20E+05 1.20E−04 1.30E−10 AB274 IL-17 (SEQ 2) 1.10E+06 8.20E−067.40E−12 DVD2157 IL-17 (SEQ 2) 1.50E+06 2.60E−05 1.80E−11 DVD2157 IL-13(SEQ 1) 3.30E+05 6.90E−05 2.10E−10 AB397 IL-13 (SEQ 1) 9.20E+05 1.20E−041.30E−10 AB274 IL-17 (SEQ 2) 1.10E+06 8.20E−06 7.40E−12 DVD2158 IL-13(SEQ 1) 1.60E+06 1.40E−04 8.60E−11 DVD2158 IL-17 (SEQ 2) 2.00E+04<1.0e−6     <5.0E−11  DVD2159 IL-17 (SEQ 2) 2.50E+06 1.20E−05 4.90E−12DVD2159 IL-13 (SEQ 1) 2.60E+05 5.30E−05 2.00E−10 AB397 IL-13 (SEQ 1)9.20E+05 1.20E−04 1.30E−10 AB275 IL-17 (SEQ 3) 1.90E+06 6.50E−053.40E−11 DVD2161 IL-17 (SEQ 3) 1.60E+06 4.50E−05 2.70E−11 DVD2161 IL-13(SEQ 1) 1.10E+05 4.10E−05 3.80E−10 AB397 IL-13 (SEQ 1) 9.20E+05 1.20E−041.30E−10 AB275 IL-17 (SEQ 3) 1.90E+06 6.50E−05 3.40E−11 DVD2164 IL-13(SEQ 1) 1.50E+06 1.70E−04 1.10E−10 DVD2164 IL-17 (SEQ 3) 1.80E+043.10E−05 1.70E−09 DVD2165 IL-17 (SEQ 3) 2.30E+06 3.20E−05 1.40E−11DVD2165 IL-13 (SEQ 1) 2.60E+05 5.70E−05 2.20E−10 AB398 IL-13 (SEQ 2)6.30E+05 2.80E−05 4.50E−11 AB273 IL-17 (SEQ 1) 2.00E+06 <1.0e−6    <5.0E−13  DVD2166 IL-13 (SEQ 2) 7.60E+05 2.60E−05 3.40E−11 DVD2166 IL-17(SEQ 1) 1.30E+05 9.40E−06 7.50E−11 DVD2167 IL-17 (SEQ 1) 2.70E+06<1.0e−6     <3.7E−13  DVD2167 IL-13 (SEQ 2) 2.10E+04 <1.0e−6    <4.8e−11   AB398 IL-13 (SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB273 IL-17(SEQ 1) 2.00E+06 <1.0e−6     <5.0E−13  DVD2168 IL-13 (SEQ 2) 6.80E+053.70E−05 5.40E−11 DVD2168 IL-17 (SEQ 1) 4.00E+05 <1.0e−6     <2.5E−12 AB398 IL-13 (SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB273 IL-17 (SEQ 1)2.00E+06 <1.0e−6     <5.0E−13  DVD2171 IL-17 (SEQ 1) 1.90E+06 2.10E−051.10E−11 DVD2171 IL-13 (SEQ 2) 2.00E+05 <1.0e−6     <5e−12 AB398 IL-13(SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB274 IL-17 (SEQ 2) 1.10E+06 8.20E−067.40E−12 DVD2172 IL-13 (SEQ 2) 8.90E+05 2.90E−05 3.30E−11 DVD2172 IL-17(SEQ 2) 6.80E+03 1.70E−04 2.60E−08 DVD2173 IL-17 (SEQ 2) 2.50E+069.00E−06 3.60E−12 DVD2173 IL-13 (SEQ 2) 3.10E+05 3.70E−05 1.20E−10 AB398IL-13 (SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB274 IL-17 (SEQ 2) 1.10E+068.20E−06 7.40E−12 DVD2174 IL-13 (SEQ 2) 7.90E+05 2.80E−05 3.60E−11DVD2174 IL-17 (SEQ 2) 1.80E+05 1.80E−05 9.80E−11 DVD2175 IL-17 (SEQ 2)2.70E+06 5.40E−06 2.10E−12 DVD2175 IL-13 (SEQ 2) 5.20E+04 <1.0e−6    <1.9e−11   AB398 IL-13 (SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB274 IL-17(SEQ 2) 1.10E+06 8.20E−06 7.40E−12 DVD2177 IL-17 (SEQ 2) 2.50E+061.00E−05 4.30E−12 DVD2177 IL-13 (SEQ 2) 1.10E+05 <1.0e−6     <9.1e−12  AB398 IL-13 (SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB275 IL-17 (SEQ 3)1.90E+06 6.50E−05 3.40E−11 DVD2179 IL-17 (SEQ 3) 1.70E+06 6.40E−053.90E−11 DVD2179 IL-13 (SEQ 2) 2.80E+05 1.00E−06 3.60E−12 AB398 IL-13(SEQ 2) 6.30E+05 2.80E−05 4.50E−11 AB275 IL-17 (SEQ 3) 1.90E+06 6.50E−053.40E−11 DVD2181 IL-17 (SEQ 3) 2.60E+06 4.20E−05 1.60E−11 DVD2181 IL-13(SEQ 2) 8.20E+04 <1.0e−6     <1.2e−11   AB398 IL-13 (SEQ 2) 6.30E+052.80E−05 4.50E−11 AB275 IL-17 (SEQ 3) 1.90E+06 6.50E−05 3.40E−11 DVD2183IL-17 (SEQ 3) 2.30E+06 3.60E−05 1.50E−11 DVD2183 IL-13 (SEQ 2) 2.40E+05<1.0e−6     <4.2e−12   AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11AB273 IL-17 (SEQ 1) 2.00E+06 <1.0e−6     <5.0E−13  DVD2185 IL-17 (SEQ 1)1.70E+06 1.50E−05 8.90E−12 DVD2185 IL-13 (SEQ 3) 1.10E+05 <1.0e−6    <9.1e−12   AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11 AB273 IL-17(SEQ 1) 2.00E+06 <1.0e−6     <5.0E−13  DVD2187 IL-17 (SEQ 1) 2.60E+06<1.0e−6     <3.8E−13  DVD2187 IL-13 (SEQ 3) 2.90E+05 2.10E−05 7.20E−11AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11 AB273 IL-17 (SEQ 1)2.00E+06 <1.0e−6     <5.0E−13  DVD2189 IL-17 (SEQ 1) 2.50E+06<1.0e−6     <4.0E−13  DVD2189 IL-13 (SEQ 3) 4.10E+05 3.90E−05 9.40E−11AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11 AB274 IL-17 (SEQ 2)1.10E+06 8.20E−06 7.40E−12 DVD2190 IL-13 (SEQ 3) 2.60E+06 5.70E−052.10E−11 DVD2190 IL-17 (SEQ 2) 8.90E+04 3.40E−06 3.80E−11 DVD2191 IL-17(SEQ 2) 2.50E+06 1.20E−05 4.60E−12 DVD2191 IL-13 (SEQ 3) 2.60E+054.40E−05 1.70E−10 AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11 AB274IL-17 (SEQ 2) 1.10E+06 8.20E−06 7.40E−12 DVD2192 IL-13 (SEQ 3) 1.50E+065.10E−05 3.50E−11 DVD2192 IL-17 (SEQ 2) 2.60E+05 <1.0e−6     <3.8E−12 DVD2193 IL-17 (SEQ 2) 1.90E+06 2.20E−05 1.20E−11 DVD2193 IL-13 (SEQ 3)5.90E+05 3.90E−05 6.60E−11 AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−051.90E−11 AB274 IL-17 (SEQ 2) 1.10E+06 8.20E−06 7.40E−12 DVD2195 IL-17(SEQ 2) 2.40E+06 4.80E−06 2.00E−12 DVD2195 IL-13 (SEQ 3) 2.60E+063.70E−05 1.40E−11 AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11 AB275IL-17 (SEQ 3) 1.90E+06 6.50E−05 3.40E−11 DVD2196 IL-13 (SEQ 3) 2.20E+066.40E−05 2.90E−11 DVD2196 IL-17 (SEQ 3) 1.10E+05 <1.0e−6     <9.1E−12 DVD2197 IL-17 (SEQ 3) 1.60E+06 6.20E−05 3.80E−11 DVD2197 IL-13 (SEQ 3)3.10E+05 4.40E−05 1.40E−10 AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−051.90E−11 AB275 IL-17 (SEQ 3) 1.90E+06 6.50E−05 3.40E−11 DVD2199 IL-17(SEQ 3) 2.30E+06 4.40E−05 1.90E−11 DVD2199 IL-13 (SEQ 3) 1.30E+063.40E−05 2.60E−11 AB399 IL-13 (SEQ 3) 3.40E+06 6.40E−05 1.90E−11 AB275IL-17 (SEQ 3) 1.90E+06 6.50E−05 3.40E−11 DVD2200 IL-13 (SEQ 3) 1.30E+066.00E−05 4.50E−11 DVD2200 IL-17 (SEQ 3) 1.80E+05 4.60E−06 2.50E−11DVD2201 IL-17 (SEQ 3) 1.60E+06 6.30E−05 4.00E−11 DVD2201 IL-13 (SEQ 3)2.40E+06 5.70E−05 2.40E−11

Binding of all DVD-Ig proteins characterized by Biacore technology wasmaintained and comparable to that of parent antibodies. All variabledomains bound with similar high affinity as the parent antibodies.

Example 4 Characterization of Antibodies and DVD-Ig Proteins

The ability of purified DVD-Ig protein to inhibit a functional activitywas determined, e.g., using the cytokine bioassay as described inExample 3. The binding affinities of the DVD-Ig protein to recombinanthuman antigen were determined using surface plasmon resonance (Biacore®)measurement as described in Example 4. The IC₅₀ values from thebioassays and the affinity of the antibodies and DVD-Ig proteins wereranked. The DVD-Ig protein that fully maintain the activity of theparent mAbs were selected as candidates for future development. The top2-3 most favorable DVD-Ig proteins were further characterized.

Example 4.1 Pharmacokinetic Analysis of Humanized Antibodies or DVD-IgProtein

Pharmacokinetic studies are carried out in Sprague-Dawley rats andcynomolgus monkeys. Male and female rats and cynomolgus monkeys aredosed intravenously or subcutaneously with a single dose of 4 mg/kg mAbor DVD-Ig protein and samples are analyzed using antigen capture ELISA,and pharmacokinetic parameters are determined by noncompartmentalanalysis. Briefly, ELISA plates are coated with goat anti-biotinantibody (5 mg/ml, 4° C., overnight), blocked with Superblock (Pierce),and incubated with biotinylated human antigen at 50 ng/ml in 10%Superblock TTBS at room temperature for 2 hours. Serum samples areserially diluted (0.5% serum, 10% Superblock in TTBS) and incubated onthe plate for 30 minutes at room temperature. Detection is carried outwith HRP-labeled goat anti human antibody and concentrations aredetermined with the help of standard curves using the four parameterlogistic fit. Values for the pharmacokinetic parameters are determinedby non-compartmental model using WinNonlin software (PharsightCorporation, Mountain View, Calif.). Humanized mAbs with goodpharmacokinetics profile (T½ is 8-13 days or better, with low clearanceand excellent bioavailability 50-100%) are selected.

Example 4.2 Physicochemical and In Vitro Stability Analysis of HumanizedMonoclonal Antibodies and DVD-Ig Proteins Size Exclusion Chromatography

Antibodies or DVD-Ig proteins were diluted to 2.5 mg/mL with water and20 mL was analyzed on a Shimadzu HPLC system using a TSK gel G3000 SWXLcolumn (Tosoh Bioscience, cat# k5539-05k). Samples were eluted from thecolumn with 211 mM sodium sulfate, 92 mM sodium phosphate, pH 7.0, at aflow rate of 0.3 mL/minutes. The HPLC system operating conditions wereas follows:

Mobile phase: 211 mM Na₂SO₄, 92 mM Na₂HPO₄*7H₂O, pH 7.0

Gradient: Isocratic

Flow rate: 0.3 mL/minute

Detector wavelength: 280 nm

Autosampler cooler temp: 4° C.

Column oven temperature: Ambient

Run time: 50 minutes

Table 10 contains purity data of parent antibodies and DVD-Ig proteinsexpressed as percent monomer (unaggregated protein of the expectedmolecular weight) as determined by the above protocol.

TABLE 10 Purity of Parent Antibodies and DVD-Ig Proteins as Determinedby Size Exclusion Chromatography Parent N-terminal C-terminal Antibodyor Variable Domain Variable Domain % Monomer DVD-Ig ID (VD) (VD)(purity) AB273 IL-17 (SEQ 1) 100 AB274 IL-17 (SEQ 2) 70.7 AB275 IL-17(SEQ 3) 94.5 AB397 IL-13 (SEQ 1) 93 AB398 IL-13 (SEQ 2) 92.8 AB399 IL-13(SEQ 3) 98.8 DVD2148 IL-13 (SEQ 1) IL-17 (SEQ 1) 69.6 DVD2150 IL-13(SEQ 1) IL-17 (SEQ 1) 68.7 DVD2152 IL-13 (SEQ 1) IL-17 (SEQ 1) 63.5DVD2153 IL-17 (SEQ 1) IL-13 (SEQ 1) 79 DVD2154 IL-13 (SEQ 1) IL-17 (SEQ2) 78.3 DVD2155 IL-17 (SEQ 2) IL-13 (SEQ 1) 93.2 DVD2156 IL-13 (SEQ 1)IL-17 (SEQ 2) 75.1 DVD2157 IL-17 (SEQ 2) IL-13 (SEQ 1) 92.5 DVD2158IL-13 (SEQ 1) IL-17 (SEQ 2) 81.4 DVD2159 IL-17 (SEQ 2) IL-13 (SEQ 1)94.9 DVD2160 IL-13 (SEQ 1) IL-17 (SEQ 3) 73.1 DVD2161 IL-17 (SEQ 3)IL-13 (SEQ 1) 85.6 DVD2162 IL-13 (SEQ 1) IL-17 (SEQ 3) 66 DVD2163 IL-17(SEQ 3) IL-13 (SEQ 1) 70 DVD2164 IL-13 (SEQ 1) IL-17 (SEQ 3) 84 DVD2165IL-17 (SEQ 3) IL-13 (SEQ 1) 84 DVD2166 IL-13 (SEQ 2) IL-17 (SEQ 1) 82.5DVD2167 IL-17 (SEQ 1) IL-13 (SEQ 2) 94 DVD2168 IL-13 (SEQ 2) IL-17(SEQ 1) 94 DVD2169 IL-17 (SEQ 1) IL-13 (SEQ 2) 68.5 DVD2170 IL-13 (SEQ2) IL-17 (SEQ 1) 73 DVD2171 IL-17 (SEQ 1) IL-13 (SEQ 2) 83 DVD2172 IL-13(SEQ 2) IL-17 (SEQ 2) 82 DVD2173 IL-17 (SEQ 2) IL-13 (SEQ 2) 91 DVD2174IL-13 (SEQ 2) IL-17 (SEQ 2) 83 DVD2175 IL-17 (SEQ 2) IL-13 (SEQ 2) 88DVD2176 IL-13 (SEQ 2) IL-17 (SEQ 2) 78 DVD2177 IL-17 (SEQ 2) IL-13 (SEQ2) 94 DVD2178 IL-13 (SEQ 2) IL-17 (SEQ 3) 65 DVD2179 IL-17 (SEQ 3) IL-13(SEQ 2) 81 DVD2180 IL-13 (SEQ 2) IL-17 (SEQ 3) 69 DVD2181 IL-17 (SEQ 3)IL-13 (SEQ 2) 88 DVD2182 IL-13 (SEQ 2) IL-17 (SEQ 3) 62 DVD2183 IL-17(SEQ 3) IL-13 (SEQ 2) 89 DVD2185 IL-17 (SEQ 1) IL-13 (SEQ 3) 99.9DVD2187 IL-17 (SEQ 1) IL-13 (SEQ 3) 84 DVD2189 IL-17 (SEQ 1) IL-13 (SEQ3) 84 DVD2190 IL-13 (SEQ 3) IL-17 (SEQ 2) 99 DVD2191 IL-17 (SEQ 2) IL-13(SEQ 3) 98 DVD2192 IL-13 (SEQ 3) IL-17 (SEQ 2) 99 DVD2193 IL-17 (SEQ 2)IL-13 (SEQ 3) 99 DVD2195 IL-17 (SEQ 2) IL-13 (SEQ 3) 96 DVD2196 IL-13(SEQ 3) IL-17 (SEQ 3) 99 DVD2197 IL-17 (SEQ 3) IL-13 (SEQ 3) 96 DVD2199IL-17 (SEQ 3) IL-13 (SEQ 3) 93 DVD2200 IL-13 (SEQ 3) IL-17 (SEQ 3) 92DVD2201 IL-17 (SEQ 3) IL-13 (SEQ 3) 99 DVD-Ig proteins showed anexcellent SEC profile with most DVD-Ig proteins showing >90% monomer.This DVD-Ig protein profile was similar to that observed for parentantibodies.

SDS-PAGE

Antibodies and DVD-Ig proteins are analyzed by sodium dodecylsulfate—polyacrylamide gel electrophoresis (SDS-PAGE) under bothreducing and non-reducing conditions. Adalimumab lot AFP04C is used as acontrol. For reducing conditions, the samples are mixed 1:1 with 2× trisglycine SDS-PAGE sample buffer (Invitrogen, cat# LC2676, lot#1323208)with 100 mM DTT, and heated at 60° C. for 30 minutes. For non-reducingconditions, the samples are mixed 1:1 with sample buffer and heated at100° C. for 5 minutes. The reduced samples (10 mg per lane) are loadedon a 12% pre-cast tris-glycine gel (Invitrogen, cat# EC6005box,lot#6111021), and the non-reduced samples (10 mg per lane) are loaded onan 8%-16% pre-cast tris-glycine gel (Invitrogen, cat# EC6045box,lot#6111021). SeeBlue Plus 2 (Invitrogen, cat#LC5925, lot#1351542) isused as a molecular weight marker. The gels are run in a XCell SureLockmini cell gel box (Invitrogen, cat#E10001) and the proteins areseparated by first applying a voltage of 75 to stack the samples in thegel, followed by a constant voltage of 125 until the dye front reachedthe bottom of the gel. The running buffer used is 1× tris glycine SDSbuffer, prepared from a 10× tris glycine SDS buffer (ABC,MPS-79-080106)). The gels are stained overnight with colloidal bluestain (Invitrogen cat#46-7015, 46-7016) and destained with Milli-Q wateruntil the background is clear. The stained gels are then scanned usingan Epson Expression scanner (model 1680, S/N DASX003641).

Sedimentation Velocity Analysis

Antibodies or DVD-Ig proteins are loaded into the sample chamber of eachof three standard two-sector carbon epon centerpieces. Thesecenterpieces have a 1.2 cm optical path length and are built withsapphire windows. PBS is used for a reference buffer and each chambercontained 140 μL. All samples are examined simultaneously using a 4-hole(AN-60Ti) rotor in a Beckman ProteomeLab XL-I analytical ultracentrifuge(serial #PL106C01).

Run conditions are programmed and centrifuge control is performed usingProteomeLab (v5.6). The samples and rotor are allowed to thermallyequilibrate for one hour prior to analysis (20.0±0.1° C.). Confirmationof proper cell loading is performed at 3000 rpm and a single scan isrecorded for each cell. The sedimentation velocity conditions are thefollowing:

Sample Cell Volume: 420 mL

Reference Cell Volume: 420 mL

Temperature: 20° C.

Rotor Speed: 35,000 rpm

Time: 8:00 hours

UV Wavelength: 280 nm

Radial Step Size: 0.003 cm

Data Collection: One data point per step without signal averaging.

Total Number of Scans: 100

LC-MS Molecular Weight Measurement of Intact Antibodies

Molecular weight of intact antibodies and DVD-Ig proteins are analyzedby LC-MS. Each antibody or DVD-Ig protein is diluted to approximately 1mg/mL with water. An 1100 HPLC (Agilent) system with a protein microtrap(Michrom Bioresources, Inc, cat#004/25109/03) is used to desalt andintroduce 5 mg of the sample into an API Qstar pulsar i massspectrometer (Applied Biosystems). A short gradient is used to elute thesamples. The gradient is run with mobile phase A (0.08% FA, 0.02% TFA inHPLC water) and mobile phase B (0.08% FA and 0.02% TFA in acetonitrile)at a flow rate of 50 mL/minute. The mass spectrometer is operated at 4.5kvolts spray voltage with a scan range from 2000 to 3500 mass to chargeratio.

LC-MS Molecular Weight Measurement of Antibody and DVD-Ig Protein Lightand Heavy Chains

Molecular weight measurement of antibody and DVD-Ig protein light chain(LC), heavy chain (HC) and deglycosylated HC are analyzed by LC-MS.Antibodies and DVD-Ig proteins are diluted to 1 mg/mL with water and thesample is reduced to LC and HC with a final concentration of 10 mM DTTfor 30 minutes at 37° C. To deglycosylate the antibodies and DVD-Igproteins, 100 mg of the antibody or DVD-Ig protein is incubated with 2mL of PNGase F, 5 mL of 10% N-octylglucoside in a total volume of 100 mLovernight at 37° C. After deglycosylation the sample is reduced with afinal concentration of 10 mM DTT for 30 minutes at 37° C. An Agilent1100 HPLC system with a C4 column (Vydac, cat#214TP5115, S/N060206537204069) is used to desalt and introduce the sample (5 mg) intoan API Qstar pulsar i mass spectrometer (Applied Biosystems). A shortgradient is used to elute the sample. The gradient is run with mobilephase A (0.08% FA, 0.02% TFA in HPLC water) and mobile phase B (0.08% FAand 0.02% TFA in acetonitrile) at a flow rate of 50 mL/minute. The massspectrometer is operated at 4.5 kvolts spray voltage with a scan rangefrom 800 to 3500 mass to charge ratio.

Peptide Mapping

The antibody or DVD-Ig protein is denatured for 15 minutes at roomtemperature with a final concentration of 6 M guanidine hydrochloride in75 mM ammonium bicarbonate. The denatured samples are reduced with afinal concentration of 10 mM DTT at 37° C. for 60 minutes, followed byalkylation with 50 mM iodoacetic acid (IAA) in the dark at 37° C. for 30minutes. Following alkylation, the sample is dialyzed overnight againstfour liters of 10 mM ammonium bicarbonate at 4° C. The dialyzed sampleis diluted to 1 mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mgof antibody or DVD-Ig protein is either digested with trypsin (Promega,cat#V5111) or Lys-C (Roche, cat#11 047 825 001) at a 1:20 (w/w)trypsin/Lys-C:antibody or DVD-Ig protein ratio at 37° C. for 4 hours.Digests are quenched with 1 mL of 1 N HCl. For peptide mapping with massspectrometer detection, 40 mL of the digests are separated by reversephase high performance liquid chromatography (RPHPLC) on a C18 column(Vydac, cat#218TP51, S/N NE9606 10.3.5) with an Agilent 1100 HPLCsystem. The peptide separation is run with a gradient using mobile phaseA (0.02% TFA and 0.08% FA in HPLC grade water) and mobile phase B (0.02%TFA and 0.08% FA in acetonitrile) at a flow rate of 50 mL/minutes. TheAPI QSTAR Pulsar i mass spectromer is operated in positive mode at 4.5kvolts spray voltage and a scan range from 800 to 2500 mass to chargeratio.

Disulfide Bond Mapping

To denature the antibody, 100 mL of the antibody or DVD-Ig protein ismixed with 300 mL of 8 M guanidine HCl in 100 mM ammonium bicarbonate.The pH is checked to ensure that it is between 7 and 8 and the samplesare denatured for 15 minutes at room temperature in a finalconcentration of 6 M guanidine HCl. A portion of the denatured sample(100 mL) is diluted to 600 mL with Milli-Q water to give a finalguanidine-HCl concentration of 1 M. The sample (220 mg) is digested witheither trypsin (Promega, cat #V5111, lot#22265901) or Lys-C (Roche,cat#11047825001, lot#12808000) at a 1:50 trypsin or 1:50 Lys-C: antibodyor DVD-Ig protein (w/w) ratios (4.4 mg enzyme: 220 mg sample) at 37° C.for approximately 16 hours. An additional 5 mg of trypsin or Lys-C isadded to the samples and digestion is allowed to proceed for anadditional 2 hours at 37° C. Digestions are stopped by adding 1 mL ofTFA to each sample. Digested samples are separated by RPHPLC using a C18column (Vydac, cat#218TP51 S/N NE020630-4-1A) on an Agilent HPLC system.The separation is run with the same gradient used for peptide mappingusing mobile phase A (0.02% TFA and 0.08% FA in HPLC grade water) andmobile phase B (0.02% TFA and 0.08% FA in acetonitrile) at a flow rateof 50 mL/minute. The HPLC operating conditions are the same as thoseused for peptide mapping. The API QSTAR Pulsar i mass spectromer isoperated in positive mode at 4.5 kvolts spray voltage and a scan rangefrom 800 to 2500 mass-to-charge ratio. Disulfide bonds are assigned bymatching the observed MWs of peptides with the predicted MWs of trypticor Lys-C peptides linked by disulfide bonds.

Free Sulfhydryl Determination

The method used to quantify free cysteines in an antibody or DVD-Igprotein is based on the reaction of Ellman's reagent,5,50-dithio-bis(2-nitrobenzoic acid) (DTNB), with sulfhydryl groups (SH)which gives rise to a characteristic chromophoric product,5-thio-(2-nitrobenzoic acid) (TNB). The reaction is illustrated in theformula:

DTNB+RSH®RS−TNB+TNB−+H+

The absorbance of the TNB− is measured at 412 nm using a Cary 50spectrophotometer. An absorbance curve is plotted using dilutions of 2mercaptoethanol (b-ME) as the free SH standard and the concentrations ofthe free sulfhydryl groups in the protein are determined from absorbanceat 412 nm of the sample.

The b-ME standard stock is prepared by a serial dilution of 14.2 M b-MEwith HPLC grade water to a final concentration of 0.142 mM. Thenstandards in triplicate for each concentration are prepared. Antibody orDVD-Ig protein is concentrated to 10 mg/mL using an amicon ultra 10,000MWCO centrifugal filter (Millipore, cat# UFC801096, lot# L3KN5251) andthe buffer is changed to the formulation buffer used for adalimumab(5.57 mM sodium phosphate monobasic, 8.69 mM sodium phosphate dibasic,106.69 mM NaCl, 1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mMmannitol, pH 5.2, 0.1% (w/v) Tween). The samples are mixed on a shakerat room temperature for 20 minutes. Then 180 mL of 100 mM Tris buffer,pH 8.1 is added to each sample and standard followed by the addition of300 mL of 2 mM DTNB in 10 mM phosphate buffer, pH 8.1. After thoroughmixing, the samples and standards are measured for absorption at 412 nmon a Cary 50 spectrophotometer. The standard curve is obtained byplotting the amount of free SH and OD₄₁₂ nm of the b-ME standards. FreeSH content of samples are calculated based on this curve aftersubtraction of the blank.

Weak Cation Exchange Chromatography

Antibody or DVD-Ig protein is diluted to 1 mg/mL with 10 mM sodiumphosphate, pH 6.0. Charge heterogeneity is analyzed using a ShimadzuHPLC system with a WCX-10 ProPac analytical column (Dionex, cat#054993,S/N 02722). The samples are loaded on the column in 80% mobile phase A(10 mM sodium phosphate, pH 6.0) and 20% mobile phase B (10 mM sodiumphosphate, 500 mM NaCl, pH 6.0) and eluted at a flow rate of 1.0mL/minute.

Oligosaccharide Profiling

Oligosaccharides released after PNGase F treatment of antibody or DVD-Igprotein are derivatized with 2-aminobenzamide (2-AB) labeling reagent.The fluorescent-labeled oligosaccharides are separated by normal phasehigh performance liquid chromatography (NPHPLC) and the different formsof oligosaccharides are characterized based on retention time comparisonwith known standards.

The antibody or DVD-Ig protein is first digested with PNGaseF to cleaveN-linked oligosaccharides from the Fc portion of the heavy chain. Theantibody or DVD-Ig protein (200 mg) is placed in a 500 mL Eppendorf tubealong with 2 mL PNGase F and 3 mL of 10% N-octylglucoside. Phosphatebuffered saline is added to bring the final volume to 60 mL. The sampleis incubated overnight at 37° C. in an Eppendorf thermomixer set at 700RPM. Adalimumab lot AFP04C is also digested with PNGase F as a control.

After PNGase F treatment, the samples are incubated at 95° C. for 5minutes in an Eppendorf thermomixer set at 750 RPM to precipitate outthe proteins, then the samples are placed in an Eppendorf centrifuge for2 minutes at 10,000 RPM to spin down the precipitated proteins. Thesupernatent containing the oligosaccharides are transferred to a 500 mLEppendorf tube and dried in a speed-vac at 65° C.

The oligosaccharides are labeled with 2AB using a 2AB labeling kitpurchased from Prozyme (cat# GKK-404, lot#132026). The labeling reagentis prepared according to the manufacturer's instructions. Acetic acid(150 mL, provided in kit) is added to the DMSO vial (provided in kit)and mixed by pipeting the solution up and down several times. The aceticacid/DMSO mixture (100 mL) is transferred to a vial of 2-AB dye (justprior to use) and mixed until the dye is fully dissolved. The dyesolution is then added to a vial of reductant (provided in kit) andmixed well (labeling reagent). The labeling reagent (5 mL) is added toeach dried oligosaccharide sample vial, and mixed thoroughly. Thereaction vials are placed in an Eppendorf thermomixer set at 65° C. and700-800 RPM for 2 hours of reaction.

After the labeling reaction, the excess fluorescent dye is removed usingGlycoClean S Cartridges from Prozyme (cat# GKI-4726). Prior to addingthe samples, the cartridges are washed with 1 mL of milli-Q waterfollowed with 5 ishes of 1 mL 30% acetic acid solution. Just prior toadding the samples, 1 mL of acetonitrile (Burdick and Jackson, cat#AH015-4) is added to the cartridges.

After all of the acetonitrile passed through the cartridge, the sampleis spotted onto the center of the freshly washed disc and allowed toadsorb onto the disc for 10 minutes. The disc is washed with 1 mL ofacetonitrile followed by five ishes of 1 mL of 96% acetonitrile. Thecartridges are placed over a 1.5 mL Eppendorf tube and the 2-AB labeledoligosaccharides are eluted with 3 ishes (400 mL each ish) of milli Qwater.

The oligosaccharides are separated using a Glycosep N HPLC (cat#GKI-4728) column connected to a Shimadzu HPLC system. The Shimadzu HPLCsystem consisted of a system controller, degasser, binary pumps,autosampler with a sample cooler, and a fluorescent detector.

Stability at Elevated Temperatures

The buffer of antibody or DVD-Ig protein is either 5.57 mM sodiumphosphate monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl,1.07 mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, 0.1%(w/v) Tween, pH 5.2; or mM histidine, 10 mM methionine, 4% mannitol, pH5.9 using Amicon ultra centrifugal filters. The final concentration ofthe antibodies or DVD-Ig proteins is adjusted to 2 mg/mL with theappropriate buffers. The antibody or DVD-Ig protein solutions are thenfilter sterized and 0.25 mL aliquots are prepared under sterileconditions. The aliquots are left at either −80° C., 5° C., 25° C., or40° C. for 1, 2 or 3 weeks. At the end of the incubation period, thesamples are analyzed by size exclusion chromatography and SDS-PAGE.

The stability samples are analyzed by SDS-PAGE under both reducing andnon-reducing conditions. The procedure used is the same as describedherein. The gels are stained overnight with colloidal blue stain(Invitrogen cat#46-7015, 46-7016) and destained with Milli-Q water untilthe background is clear. The stained gels are then scanned using anEpson Expression scanner (model 1680, S/N DASX003641). To obtain moresensitivity, the same gels are silver stained using silver staining kit(Owl Scientific) and the recommended procedures given by themanufacturer is used.

Dynamic Scanning Fluorimetry

The DVD-Igs proteins were dialysed in 10 mM citrate 10 mM phosphatebuffer, pH 6.0 to get a final concentration of 1 mg/ml. Triplicates wererun for each DVD-Ig protein. For each sample, 27 μl of the DVD-Igprotein was added in a well of a 96 well plate and mixed with 3 μl of 4×diluted SYPRO Orange dye (Invitrogen). The dye is supplied in DMSO at aconcentration of 5000× and was diluted to the working concentration of4× in water. The plate was centrifuged for 30 seconds to ensure thatboth the dye and the protein settle to the bottom of the wells andcomplete mixing was ensured by gentle aspiration by a pipette tip. Theplate was then sealed with an adhesive film.

A real time PCR (Applied Biosciences, 7500 Series) was used formeasuring the change in fluorescence intensities with temperature. Theplate was heated from 25° C. to 95° C. at a temperature ramp rate ofapproximately 0.5° C./minute and emission fluorescence was collectedusing TAMRA filter. The data was exported to Microsoft Excel and plottedas temperature vs fluorescence for each DVD-Ig protein. Onset of meltingwas noted as the temperature where the thermogram rises above thebaseline fluorescence. SYPRO Orange is a hydrophobic dye andpreferentially binds to the exposed hydrophobic residues in an unfoldedprotein molecule. Hence the onset of unfolding temperature, as measuredby an increase in fluorescence, is an indication of the thermalstability of the DVD-Ig protein. The unfolding temperature for theDVD-Ig proteins can be found in Table 11.

TABLE 11 Thermal Stability of DVD-Ig Proteins as Determined by DynamicScanning Fluorimetry Parent Antibody N-terminal C-terminal or DVD-Variable Variable T onset Ig ID Domain (VD) Domain (VD) (deg C.) DVD2148IL-13 (SEQ 1) IL-17 (SEQ 1) 62 DVD2150 IL-13 (SEQ 1) IL-17 (SEQ 1) 61DVD2152 IL-13 (SEQ 1) IL-17 (SEQ 1) 60.5 DVD2154 IL-13 (SEQ 1) IL-17(SEQ 2) 61.5 DVD2155 IL-17 (SEQ 2) IL-13 (SEQ 1) DVD2156 IL-13 (SEQ 1)IL-17 (SEQ 2) 59 DVD2157 IL-17 (SEQ 2) IL-13 (SEQ 1) 50.5 DVD2158 IL-13(SEQ 1) IL-17 (SEQ 2) 63 DVD2159 IL-17 (SEQ 2) IL-13 (SEQ 1) 49.5DVD2160 IL-13 (SEQ 1) IL-17 (SEQ 3) 64 DVD2161 IL-17 (SEQ 3) IL-13(SEQ 1) 48 DVD2162 IL-13 (SEQ 1) IL-17 (SEQ 3) 60.5 DVD2163 IL-17 (SEQ3) IL-13 (SEQ 1) 48 DVD2164 IL-13 (SEQ 1) IL-17 (SEQ 3) 61.5 DVD2165IL-17 (SEQ 3) IL-13 (SEQ 1) 50 DVD2166 IL-13 (SEQ 2) IL-17 (SEQ 1) 62.5DVD2167 IL-17 (SEQ 1) IL-13 (SEQ 2) 50.5 DVD2168 IL-13 (SEQ 2) IL-17(SEQ 1) 46 DVD2170 IL-13 (SEQ 2) IL-17 (SEQ 1) 61.5 DVD2172 IL-13 (SEQ2) IL-17 (SEQ 2) 63.5 DVD2173 IL-17 (SEQ 2) IL-13 (SEQ 2) 55.5 DVD2174IL-13 (SEQ 2) IL-17 (SEQ 2) 64.5 DVD2175 IL-17 (SEQ 2) IL-13 (SEQ 2) 53DVD2176 IL-13 (SEQ 2) IL-17 (SEQ 2) 64 DVD2177 IL-17 (SEQ 2) IL-13 (SEQ2) 52.5 DVD2178 IL-13 (SEQ 2) IL-17 (SEQ 3) 64 DVD2179 IL-17 (SEQ 3)IL-13 (SEQ 2) 49.5 DVD2180 IL-13 (SEQ 2) IL-17 (SEQ 3) 62 DVD2182 IL-13(SEQ 2) IL-17 (SEQ 3) 63 DVD2183 IL-17 (SEQ 3) IL-13 (SEQ 2) 48 DVD2185IL-17 (SEQ 1) IL-13 (SEQ 3) 54.5 DVD2190 IL-13 (SEQ 3) IL-17 (SEQ 2) 48DVD2191 IL-17 (SEQ 2) IL-13 (SEQ 3) 54.5 DVD2193 IL-17 (SEQ 2) IL-13(SEQ 3) 51 DVD2195 IL-17 (SEQ 2) IL-13 (SEQ 3) 51.5 DVD2196 IL-13 (SEQ3) IL-17 (SEQ 3) 48.5 DVD2197 IL-17 (SEQ 3) IL-13 (SEQ 3) 49 DVD2199IL-17 (SEQ 3) IL-13 (SEQ 3) 45 DVD2201 IL-17 (SEQ 3) IL-13 (SEQ 3) 46

Most DVD-Ig proteins showed an unfolding temperature>50. This DVD-Igprotein profile is similar to that observed for parent antibodies.

Solubility Determination

DVD-Ig protein candidates were dialyzed in 15 mM His, pH 6.0. This wasfollowed by concentrating them upto 50 μl in centricons with a 30Kcutoff. Solubility was visually confirmed by absence of precipitationafter storage at 4° C. and quantitatively determined by UV absorbancemeasurement at 280 nm.

TABLE 12 Solubility of DVD-Ig Proteins Parent Antibody N-terminalC-terminal or DVD- Variable Variable Visual Solubility Ig ID Domain (VD)Domain (VD) observation (mg/mL) DVD2148 IL-13 (SEQ 1) IL-17 (SEQ 1)Clear >53.0 DVD2150 IL-13 (SEQ 1) IL-17 (SEQ 1) Clear >28.6 DVD2152IL-13 (SEQ 1) IL-17 (SEQ 1) Clear >57.3 DVD2154 IL-13 (SEQ 1) IL-17 (SEQ2) Clear >79.2 DVD2155 IL-17 (SEQ 2) IL-13 (SEQ 1) Ppt DVD2156 IL-13(SEQ 1) IL-17 (SEQ 2) Clear >98.9 DVD2157 IL-17 (SEQ 2) IL-13 (SEQ 1)Clear >23.2 DVD2158 IL-13 (SEQ 1) IL-17 (SEQ 2) Clear >121.6 DVD2159IL-17 (SEQ 2) IL-13 (SEQ 1) Ppt 13.3 DVD2160 IL-13 (SEQ 1) IL-17 (SEQ 3)Clear >122.3 DVD2161 IL-17 (SEQ 3) IL-13 (SEQ 1) Ppt 10.5 DVD2162 IL-13(SEQ 1) IL-17 (SEQ 3) Clear >85.2 DVD2163 IL-17 (SEQ 3) IL-13 (SEQ 1)Clear >8.6 DVD2164 IL-13 (SEQ 1) IL-17 (SEQ 3) Clear >99.1 DVD2165 IL-17(SEQ 3) IL-13 (SEQ 1) opalascent — DVD2166 IL-13 (SEQ 2) IL-17 (SEQ 1)Clear >13.8 DVD2167 IL-17 (SEQ 1) IL-13 (SEQ 2) Clear — DVD2168 IL-13(SEQ 2) IL-17 (SEQ 1) Clear — DVD2170 IL-13 (SEQ 2) IL-17 (SEQ 1) Ppt12.1 DVD2172 IL-13 (SEQ 2) IL-17 (SEQ 2) Clear >58.9 DVD2173 IL-17 (SEQ2) IL-13 (SEQ 2) phase 39.3 separation DVD2174 IL-13 (SEQ 2) IL-17 (SEQ2) Clear >75.4 DVD2175 IL-17 (SEQ 2) IL-13 (SEQ 2) Ppt DVD2176 IL-13(SEQ 2) IL-17 (SEQ 2) Clear >43.5 DVD2177 IL-17 (SEQ 2) IL-13 (SEQ 2)Clear >9.8 DVD2178 IL-13 (SEQ 2) IL-17 (SEQ 3) Clear >69.8 DVD2179 IL-17(SEQ 3) IL-13 (SEQ 2) Ppt 12.1 DVD2180 IL-13 (SEQ 2) IL-17 (SEQ 3) phase61.7 separation DVD2182 IL-13 (SEQ 2) IL-17 (SEQ 3) Clear >57.5 DVD2183IL-17 (SEQ 3) IL-13 (SEQ 2) Ppt DVD2185 IL-17 (SEQ 1) IL-13 (SEQ 3)phase separation DVD2190 IL-13 (SEQ 3) IL-17 (SEQ 2) Clear >62.4 DVD2191IL-17 (SEQ 2) IL-13 (SEQ 3) phase 44 separation DVD2193 IL-17 (SEQ 2)IL-13 (SEQ 3) Ppt DVD2195 IL-17 (SEQ 2) IL-13 (SEQ 3) Clear DVD2196IL-13 (SEQ 3) IL-17 (SEQ 3) Clear >59.8 DVD2197 IL-17 (SEQ 3) IL-13 (SEQ3) Ppt 31 DVD2199 IL-17 (SEQ 3) IL-13 (SEQ 3) opalescent DVD2201 IL-17(SEQ 3) IL-13 (SEQ 3) opalescent

Most DVD-Ig proteins showed clear appearance and could be concentratedto greater than 25 mg/ml. This DVD-Ig protein profile is similar to thatobserved for parent antibodies.

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that maybe cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein. Thedisclosure will employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

The present disclosure also incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications:

-   Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John    Wiley &Sons, NY (1993);-   Ausubel, F. M. et al. eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY    (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X);-   CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND    PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984);-   Giege, R. and Ducruix, A. Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS    AND PROTEINS, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford    University Press, New York, N.Y., (1999);-   Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, pp.    115-138 (1984);-   Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS    563-681 (Elsevier, N.Y., 1981;-   Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor    Laboratory Press, 2nd ed. 1988);-   Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST    (National Institutes of Health, Bethesda, Md. (1987) and (1991);-   Kabat, E. A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL    INTEREST, Fifth Edition, U.S. Department of Health and Human    Services, NIH Publication No. 91-3242;-   Kontermann and Dubel eds., ANTIBODY ENGINEERING (2001)    Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).-   Kriegler, Gene Transfer and Expression, A Laboratory Manual,    Stockton Press, NY (1990); Lu and Weiner eds., CLONING AND    EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS (2001) BioTechniques    Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X). MEDICAL    APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC    Pres., Boca Raton, Fla. (1974);-   Old, R. W. & S. B. Primrose, PRINCIPLES OF GENE MANIPULATION: AN    INTRODUCTION TO GENETIC ENGINEERING (3d Ed. 1985) Blackwell    Scientific Publications, Boston. Studies in Microbiology; V.2:409    pp. (ISBN 0-632-01318-4).-   Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d    Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN    0-87969-309-6). SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY    SYSTEMS, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978-   Winnacker, E. L. FROM GENES TO CLONES: INTRODUCTION TO GENE    TECHNOLOGY (1987) VCH Publishers, NY (translated by Horst    lbelgaufts). 634 pp. (ISBN 0-89573-614-4).

EQUIVALENTS

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

1-25. (canceled)
 26. A binding protein comprising first and secondpolypeptide chains, each independently comprising VD1-(X1)n-VD2-C—(X2)n,wherein VD1 is a first variable domain; VD2 is a second variable domain;C is a constant domain; X1 is a linker; X2 is an Fc region; and n is 0or 1; wherein the VD1 domains on the first and second polypeptide chainsform a first functional target binding site and the VD2 domains on thefirst and second polypeptide chains form a second functional targetbinding site, and wherein the binding protein is capable of bindingIL-13 and IL-17, wherein: (a) the variable domains that form afunctional target binding site for IL-13 independently comprise: CDRs1-3 from SEQ ID NO: 30 and CDRs 1-3 from SEQ ID NO 31, CDRs 1-3 from SEQID NO: 32 and CDRs 1-3 from SEQ ID NO 33, or CDRs 1-3 from SEQ ID NO: 34and CDRs 1-3 from SEQ ID NO 35, and the binding protein is capable ofinhibiting IL-13 with an IC₅₀ of at most about 2.442 nM, as measured bydirect bind ELISA, and/or binding IL-13 with a dissociation constant(K_(d)) of at most about 1.10×10-10 M, as measured by surface plasmonresonance; and/or (b) the variable domains that form a functional targetbinding site for IL-17 independently comprise: CDRs 1-3 from SEQ ID NO:36 and CDRs 1-3 from SEQ ID NO: 37, CDRs 1-3 from SEQ ID NO: 38 and CDRs1-3 from SEQ ID NO: 39, CDRs 1-3 from SEQ ID NO: 40 and CDRs 1-3 fromSEQ ID NO: 41, CDRs 1-3 from SEQ ID NO: 42 and CDRs 1-3 from SEQ ID NO:52, CDRs 1-3 from SEQ ID NO: 42 and CDRs 1-3 from SEQ ID NO: 53, CDRs1-3 from SEQ ID NO: 43 and CDRs 1-3 from SEQ ID NO: 50, CDRs 1-3 fromSEQ ID NO: 44 and CDRs 1-3 from SEQ ID NO: 51, CDRs 1-3 from SEQ ID NO:45 and CDRs 1-3 from SEQ ID NO: 54, CDRs 1-3 from SEQ ID NO: 46 and CDRs1-3 from SEQ ID NO: 51, CDRs 1-3 from SEQ ID NO: 47 and CDRs 1-3 fromSEQ ID NO: 54, CDRs 1-3 from SEQ ID NO: 48 and CDRs 1-3 from SEQ ID NO:51, or CDRs 1-3 from SEQ ID NO: 49 and CDRs 1-3 from SEQ ID NO: 51, andthe binding protein is capable of inhibiting IL-17 with an IC₅₀ of atmost about 0.7034 nM, as measured by direct bind ELISA, and/or (ii)binding IL-17 with a dissociation constant (K_(d)) of at most about2.60×10⁻⁸ M, as measured by surface plasmon resonance.
 27. A bindingprotein comprising first and second polypeptide chains, eachindependently comprising VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a firstvariable domain; VD2 is a second variable domain; C is a constantdomain; X1 is a linker; X2 is an Fc region; and n is 0 or 1; wherein theVD1 domains on the first and second polypeptide chains form a firstfunctional target binding site and the VD2 domains on the first andsecond polypeptide chains form a second functional target binding site,and wherein the binding protein is capable of binding IL-13 and IL-17,wherein: (a) the variable domains that form a functional target bindingsite for IL-13 independently comprise an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 30, 31, 32, 33, or 34, and thebinding protein is capable of inhibiting IL-13 with an IC₅₀ of at mostabout 2.442 nM, as measured by direct bind ELISA, and/or binding IL-13with a dissociation constant (K_(d)) of at most about 1.10×10⁻¹° M, asmeasured by surface plasmon resonance; and (b) the variable domains thatform a functional target binding site for IL-17 independently comprisean amino acid sequence selected from the group consisting of SEQ ID NOs:36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,or 54, and the binding protein is capable of inhibiting IL-17 with anIC₅₀ of at most about 0.7034 nM, as measured by direct bind ELISA,and/or binding IL-17 with a dissociation constant (K_(d)) of at mostabout 2.60×10⁻⁸ M, as measured by surface plasmon resonance.
 28. Thebinding protein according to claim 26, wherein the binding protein iscapable of binding IL-13 and IL-17, wherein (a) the variable domainsthat form a functional target binding site for IL-13 comprise SEQ IDNOs: 30 and 31, SEQ ID NOs: 32 and 33, or SEQ ID NOs: 33 and 34; and (b)the variable domains that form a functional target binding site forIL-17 comprise SEQ ID NOs: 36 and 37, SEQ ID NOs: 38 and 39, SEQ ID NOs:40 and 41, SEQ ID NOs: 42 and 52, SEQ ID NOs: 42 and 53, SEQ ID NOs: 43and 50, SEQ ID NOs: 44 and 51, SEQ ID NOs: 45 and 54, SEQ ID NOs: 46 and51, SEQ ID NOs: 47 and 54, SEQ ID NOs: 48 and 51, or SEQ ID NOs: 49 and51.
 29. The binding protein according to claim 26, wherein the firstpolypeptide chain comprises a first VD1-(X1)n-VD2-C—(X2)n, wherein VD1is a first heavy chain variable domain; VD2 is a second heavy chainvariable domain; C is a heavy chain constant domain; X1 is a linker; X2is an Fc region; and n is 0 or 1; wherein the second polypeptide chaincomprises a second VD1-(X1)n-VD2-C, wherein VD1 is a first light chainvariable domain; VD2 is a second light chain variable domain; C is alight chain constant domain; X1 is a linker; n is 1 for (X1)n; and n is0 for (X2)n wherein the VD1 domains on the first and second polypeptidechains form a first functional target binding site and the VD2 domainson the first and second polypeptide chains form a second functionaltarget binding site.
 30. The binding protein according to claim 26,comprising two first polypeptide chains and two second polypeptidechains, forming four functional target binding sites.
 31. The bindingprotein according to claim 26, wherein X1 is any one of SEQ ID NOs: 1-29or a G4S (SEQ ID NO: 29) repeating sequence.
 32. The binding proteinaccording to claim 26, wherein the Fc region is a variant sequence Fcregion.
 33. The binding protein according to claim 26, wherein the Fcregion is an Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, orIgD.
 34. The binding protein according to claim 26, wherein the bindingprotein is a crystallized binding protein.
 35. A binding protein capableof binding IL-13 and IL-17, wherein the binding protein comprises anyone of: DVD2148H (comprising SEQ ID NO: 30 and 36) and DVD2148L(comprising SEQ ID NOs: 31 and 37); DVD2149H (comprising SEQ ID NO: 36and 30) and DVD2149L (comprising SEQ ID NOs: 37 and 31); DVD2150H(comprising SEQ ID NOs: 30 and 36) and DVD2150L (comprising SEQ ID NOs:31 and 37); DVD2151H (comprising SEQ ID NOs: 36 and 30) and DVD2151L(comprising SEQ ID NOs: 37 and 31); DVD2152H (comprising SEQ ID NOs: 30and 36) and DVD2152L (comprising SEQ ID NOs: 31 and 37); DVD2153H(comprising SEQ ID NOs: 36 and 30) and DVD2153L (comprising SEQ ID NOs:37 and 31); DVD 2154H (comprising SEQ ID NOs: 30 and 38) and DVD2154L(comprising SEQ ID NOs: 31 and 39); DVD2155H (comprising SEQ ID NOs: 38and 30) and DVD2155L (comprising SEQ ID NOs: 39 and 31); DVD2156H(comprising SEQ ID NOs: 30 and 38) and DVD2156L (comprising SEQ ID NOs:31 and 39); DVD2157H (comprising SEQ ID NOs: 38 and 30) and DVD2157L(comprising SEQ ID NOs: 39 and 31); DVD2158H (comprising SEQ ID NOs: 30and 38) and DVD2158L (comprising SEQ ID NOs: 31 and 39); DVD2159H(comprising SEQ ID NOs: 38 and 30) and DVD2159L (comprising SEQ ID NOs:39 and 31); DVD2160H (comprising SEQ ID NOs: 30 and 40) and DVD2160L(comprising SEQ ID NOs: 31 and 41); DVD2161H (comprising SEQ ID NOs: 40and 30) and DVD2161L (comprising SEQ ID NOs: 41 and 31); DVD2162H(comprising SEQ ID NOs: 30 and 40) and DVD2162L (comprising SEQ ID NOs:31 and 41); DVD2163H (comprising SEQ ID NOs: 40 and 30) and DVD2163L(comprising SEQ ID NOs: 41 and 31); DVD2164H (comprising SEQ ID NOs: 30and 40) and DVD2164L (comprising SEQ ID NOs: 31 and 41); DVD2165H(comprising SEQ ID NOs: 40 and 30) and DVD2165L (comprising SEQ ID NOs:41 and 31); DVD2166H (comprising SEQ ID NOs: 32 and 36) and DVD2166L(comprising SEQ ID NOs: 33 and 37); DVD2167H (comprising SEQ ID NOs: 36and 32) and DVD2167L (comprising SEQ ID NOs: 37 and 33); DVD2168H(comprising SEQ ID NOs: 32 and 36) and DVD2168L (comprising SEQ ID NOs:33 and 37); DVD2169H (comprising SEQ ID NOs: 36 and 32) and DVD2169L(comprising SEQ ID NOs: 37 and 33); DVD2170H (comprising SEQ ID NOs: 32and 36) and DVD2170L (comprising SEQ ID NOs: 33 and 37); DVD2171H(comprising SEQ ID NOs: 36 and 32) and DVD2171L (comprising SEQ ID NOs:37 and 33); DVD2172H (comprising SEQ ID NOs: 32 and 38) and DVD2172L(comprising SEQ ID NOs: 33 and 39); DVD2173H (comprising SEQ ID NOs: 38and 32) and DVD2173L (comprising SEQ ID NOs: 39 and 33); DVD2174H(comprising SEQ ID NOs: 32 and 38) and DVD2174L (comprising SEQ ID NOs:33 and 39); DVD2175H (comprising SEQ ID NOs: 38 and 32) and DVD2175L(comprising SEQ ID NOs: 39 and 33); DVD2176H (comprising SEQ ID NOs: 32and 38) and DVD2176L (comprising SEQ ID NOs: 33 and 39); DVD2177H(comprising SEQ ID NOs: 38 and 32) and DVD2177L (comprising SEQ ID NOs:39 and 33); DVD2178H (comprising SEQ ID NOs: 32 and 40) and DVD2178L(comprising SEQ ID NOs: 33 and 41); DVD2179H (comprising SEQ ID NOs: 40and 32) and DVD2179L (comprising SEQ ID NOs: 41 and 33); DVD2180H(comprising SEQ ID NOs: 32 and 40) and DVD2180L (comprising SEQ ID NOs:33 and 41); DVD2181H (comprising SEQ ID NOs: 40 and 32) and DVD2181L(comprising SEQ ID NOs: 41 and 33); DVD2182H (comprising SEQ ID NOs: 32and 40) and DVD2182L (comprising SEQ ID NOs: 33 and 41); DVD2183H(comprising SEQ ID NOs: 40 and 32) and DVD2183L (comprising SEQ ID NOs:41 and 33); DVD2184H (comprising SEQ ID NOs: 34 and 36) and DVD2184L(comprising SEQ ID NOs: 35 and 37); DVD2185H (comprising SEQ ID NOs: 36and 34) and DVD2185L (comprising SEQ ID NOs: 37 and 35); DVD2186H(comprising SEQ ID NOs: 34 and 36) and DVD2186L (comprising SEQ ID NOs:35 and 37); DVD2187H (comprising SEQ ID NOs: 36 and 34) and DVD2187L(comprising SEQ ID NOs: 37 and 35); DVD2188H (comprising SEQ ID NOs: 34and 36) and DVD2188L (comprising SEQ ID NOs: 35 and 37); DVD2189H(comprising SEQ ID NOs: 36 and 34) and DVD2189L (comprising SEQ ID NOs:37 and 35); DVD2190H (comprising SEQ ID NOs: 34 and 38) and DVD2190L(comprising SEQ ID NOs: 35 and 39); DVD2191H (comprising SEQ ID NOs: 38and 34) and DVD2191L (comprising SEQ ID NOs: 39 and 35); DVD2192H(comprising SEQ ID NOs: 34 and 38) and DVD2192L (comprising SEQ ID NOs:35 and 39); DVD2193H (comprising SEQ ID NOs: 38 and 34) and DVD2193L(comprising SEQ ID NOs: 39 and 35); DVD2194H (comprising SEQ ID NOs: 34and 38) and DVD2194L (comprising SEQ ID NOs: 35 and 39); DVD2195H(comprising SEQ ID NOs: 38 and 34) and DVD2195L (comprising SEQ ID NOs:39 and 35); DVD2196H (comprising SEQ ID NOs: 34 and 40) and DVD2196L(comprising SEQ ID NOs: 35 and 41); DVD2197H (comprising SEQ ID NOs: 40and 34) and DVD2197L (comprising SEQ ID NOs: 41 and 35); DVD2198H(comprising SEQ ID NOs: 34 and 40) and DVD2198L (comprising SEQ ID NOs:35 and 41); DVD2199H (comprising SEQ ID NOs: 40 and 34) and DVD2199L(comprising SEQ ID NOs: 41 and 35); DVD2200H (comprising SEQ ID NOs: 34and 40) and DVD2200L (comprising SEQ ID NOs: 35 and 41); or DVD2201H(comprising SEQ ID NOs: 40 and 34) and DVD2201L (comprising SEQ ID NOs:41 and 35).
 36. A binding protein conjugate comprising a binding proteinaccording to claim 26, said binding protein conjugate further comprisingan immunoadhesion molecule, an imaging agent, a therapeutic agent, or acytotoxic agent.
 37. The binding protein conjugate of claim 36, whereinthe imaging agent is a radiolabel, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, or biotin.38. The binding protein conjugate of claim 37, wherein said radiolabelis ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.39. The binding protein conjugate of claim 36, wherein said therapeuticor cytotoxic agent is an anti-metabolite, an alkylating agent, anantibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, toxin, or an apoptotic agent. 40.An isolated nucleic acid encoding a binding protein amino acid sequenceaccording to claim
 26. 41. A vector comprising an isolated nucleic acidaccording to claim 40, wherein the vector is optionally selected fromthe group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pcDNA3.1TOPO, pEF6, pHybE, TOPO, and pBJ.
 42. A host cell comprising a vectoraccording to claim 41, wherein the host cell is optionally selected fromthe group consisting of a prokaryotic cell, a Escherichia coli cell, aeukaryotic cell, a protist cell, an animal cell, a plant cell, a fungalcell, a yeast cell, a Saccharomyces cerevisiae cell, an Sf9 cell, amammalian cell, an avian cell, an insect cell, a CHO cell, and a COScell.
 43. A method of producing a binding protein, comprising culturingthe host cell of claim 42 in culture medium under conditions sufficientto produce the binding protein.
 44. A pharmaceutical compositioncomprising the binding protein according to claim 26 and apharmaceutically acceptable carrier.
 45. The pharmaceutical compositionaccording to claim 44, further comprising at least one additionaltherapeutic agent.
 46. The pharmaceutical composition according to claim45, wherein said additional therapeutic agent is an imaging agent, acytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, aco-stimulation molecule blocker, an adhesion molecule blocker, ananti-cytokine antibody or functional fragment thereof, methotrexate,cyclosporin, rapamycin, FK506, a detectable label or reporter, a TNFantagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, or a cytokine antagonist.
 47. A method oftreating a subject for a disease or a disorder by administering thebinding protein of claim 26 to the subject, wherein the disease ordisorder is selected from the group consisting of arthritis,osteoarthritis, juvenile chronic arthritis, septic arthritis, Lymearthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy,systemic lupus erythematosus, Crohn's disease, ulcerative colitis,inflammatory bowel disease, insulin dependent diabetes mellitus,thyroiditis, asthma, allergic diseases, psoriasis, dermatitisscleroderma, graft versus host disease, organ transplant rejection,acute or chronic immune disease associated with organ transplantation,sarcoidosis, atherosclerosis, disseminated intravascular coagulation,Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatiguesyndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,microscopic vasculitis of the kidneys, chronic active hepatitis,uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia,infectious diseases, parasitic diseases, acute transverse myelitis,Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke,primary biliary cirrhosis, hemolytic anemia, malignancies, heartfailure, myocardial infarction, Addison's disease, sporadicpolyglandular deficiency type I and polyglandular deficiency type II,Schmidt's syndrome, adult (acute) respiratory distress syndrome,alopecia, alopecia greata, seronegative arthropathy, arthropathy,Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy,enteropathic synovitis, chlamydia, yersinia and salmonella associatedarthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis,atopic allergy, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Syndrome, AcquiredImmunodeficiency Related Diseases, hepatitis B, hepatitis C, commonvaried immunodeficiency (common variable hypogammaglobulinaemia),dilated cardiomyopathy, female infertility, ovarian failure, prematureovarian failure, fibrotic lung disease, cryptogenic fibrosingalveolitis, post-inflammatory interstitial lung disease, interstitialpneumonitis, connective tissue disease associated interstitial lungdisease, mixed connective tissue disease associated lung disease,systemic sclerosis associated interstitial lung disease, rheumatoidarthritis associated interstitial lung disease, systemic lupuserythematosus associated lung disease, dermatomyositis/polymyositisassociated lung disease, Sjögren's disease associated lung disease,ankylosing spondylitis associated lung disease, vasculitic diffuse lungdisease, haemosiderosis associated lung disease, drug-inducedinterstitial lung disease, fibrosis, radiation fibrosis, bronchiolitisobliterans, chronic eosinophilic pneumonia, lymphocytic infiltrativelung disease, post-infectious interstitial lung disease, goutyarthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classicalautoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKMantibody hepatitis), autoimmune mediated hypoglycaemia, type B insulinresistance with acanthosis nigricans, hypoparathyroidism, acute immunedisease associated with organ transplantation, chronic immune diseaseassociated with organ transplantation, osteoarthrosis, primarysclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathicleucopaenia, autoimmune neutropaenia, renal disease NOS,glomerulonephritides, microscopic vasulitis of the kidneys, Lymedisease, discoid lupus erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes), sympatheticophthalmia, pulmonary hypertension secondary to connective tissuedisease, Goodpasture's syndrome, pulmonary manifestation ofpolyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis,Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu'sdisease/arteritis, autoimmune thrombocytopaenia, idiopathicthrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrousautoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmunehypothyroidism, primary myxoedema, phacogenic uveitis, primaryvasculitis, vitiligo acute liver disease, chronic liver diseases,alcoholic cirrhosis, alcohol-induced liver injury, cholestasis,idiosyncratic liver disease, drug-induced hepatitis, non-alcoholicsteatohepatitis, allergy and asthma, group B streptococci (GBS)infection, mental disorders (e.g., depression and schizophrenia), Th2Type and Th1 Type mediated diseases, acute and chronic pain (differentforms of pain), and cancers such as lung, breast, stomach, bladder,colon, pancreas, ovarian, prostate and rectal cancer and hematopoieticmalignancies (leukemia and lymphoma) abetalipoproteinemia, acrocyanosis,acute and chronic parasitic or infectious processes, acute leukemia,acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acuteor chronic bacterial infection, acute pancreatitis, acute renal failure,adenocarcinomas, aerial ectopic beats, AIDS dementia complex,alcohol-induced hepatitis, allergic conjunctivitis, allergic contactdermatitis, allergic rhinitis, allograft rejection, alpha-I-antitrypsindeficiency, amyotrophic lateral sclerosis, anemia, angina pectoris,anterior horn cell degeneration, anti cd3 therapy, antiphospholipidsyndrome, anti-receptor hypersensitivity reactions, aortic andperipheral aneurysms, aortic dissection, arterial hypertension,arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation(sustained or paroxysmal), atrial flutter, atrioventricular block, Bcell lymphoma, bone graft rejection, bone marrow transplant (BMT)rejection, bundle branch block, Burkitt's lymphoma, burns, cardiacarrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy,cardiopulmonary bypass inflammation response, cartilage transplantrejection, cerebellar cortical degenerations, cerebellar disorders,chaotic or multifocal atrial tachycardia, chemotherapy associateddisorders, chronic myelocytic leukemia (CML), chronic alcoholism,chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL),chronic obstructive pulmonary disease (COPD), chronic salicylateintoxication, colorectal carcinoma, congestive heart failure,conjunctivitis, contact dermatitis, cor pulmonale, coronary arterydisease, Creutzfeldt-Jakob disease, culture negative sepsis, cysticfibrosis, cytokine therapy associated disorders, Dementia pugilistica,demyelinating diseases, dengue hemorrhagic fever, dermatitis,dermatologic conditions, diabetes, diabetes mellitus, diabeticarteriosclerotic disease, diffuse Lewy body disease, dilated congestivecardiomyopathy, disorders of the basal ganglia, Down's Syndrome inmiddle age, drug-induced movement disorders induced by drugs which blockCNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis,endocarditis, endocrinopathy, epiglottitis, Epstein-Barr virusinfection, erythromelalgia, extrapyramidal and cerebellar disorders,familial hematophagocytic lymphohistiocytosis, fetal thymus implantrejection, Friedreich's ataxia, functional peripheral arterialdisorders, fungal sepsis, gas gangrene, gastric ulcer, graft rejectionof any organ or tissue, gram negative sepsis, gram positive sepsis,granulomas due to intracellular organisms, hairy cell leukemia,Hallerrorden-Spatz disease, Hashimoto's thyroiditis, hay fever, hearttransplant rejection, hemachromatosis, hemodialysis, hemolytic uremicsyndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis A,His bundle arrhythmias, HIV infection/HIV neuropathy, Hodgkin's disease,hyperkinetic movement disorders, hypersensitivity reactions,hypersensitivity pneumonitis, hypertension, hypokinetic movementdisorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathicAddison's disease, idiopathic pulmonary fibrosis, antibody mediatedcytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammationof the aorta, influenza a, ionizing radiation exposure,iridocyclitis/uveitis/optic neuritis, ischemia-reperfusion injury,ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscularatrophy, Kaposi's sarcoma, kidney transplant rejection, legionella,leishmaniasis, leprosy, lesions of the corticospinal system, lipedema,liver transplant rejection, lymphederma, malaria, malignant lymphoma,malignant histiocytosis, malignant melanoma, meningitis,meningococcemia, metabolic/idiopathic, migraine headache, mitochondrialmulti-system disorder, mixed connective tissue disease, monoclonalgammopathy, multiple myeloma, multiple systems degenerations (Mencel,Dejerine-Thomas, Shy-Drager, or Machado-Joseph), myasthenia gravis,mycobacterium avium intracellulare, mycobacterium tuberculosis,myelodyplastic syndrome, myocardial ischemic disorders, nasopharyngealcarcinoma, neonatal chronic lung disease, nephritis, nephrosis,neurodegenerative diseases, neurogenic I muscular atrophies, neutropenicfever, non-Hodgkin's lymphoma, occlusion of the abdominal aorta and itsbranches, occlusive arterial disorders, okt3 therapy,orchitis/epidydimitis, orchitis/vasectomy reversal procedures,organomegaly, osteoporosis, pancreas transplant rejection, pancreaticcarcinoma, paraneoplastic syndrome/hypercalcemia of malignancy,parathyroid transplant rejection, pelvic inflammatory disease, perennialrhinitis, pericardial disease, peripheral atherosclerotic disease,peripheral vascular disorders, peritonitis, pernicious anemia,pneumocystis carinii pneumonia, pneumonia, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), post perfusion syndrome, post pump syndrome,post-MI cardiotomy syndrome, preeclampsia, progressive supranucleopalsy, primary pulmonary hypertension, radiation therapy, Raynaud'sphenomenon and disease, Raynoud's disease, Refsum's disease, regularnarrow QRS tachycardia, renovascular hypertension, reperfusion injury,restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, seniledementia of Lewy body type, seronegative arthropathies, shock, sicklecell anemia, skin allograft rejection, skin changes syndrome, smallbowel transplant rejection, solid tumors, specific arrhythmias, spinalataxia, spinocerebellar degenerations, streptococcal myositis,structural lesions of the cerebellum, subacute sclerosingpanencephalitis, Syncope, syphilis of the cardiovascular system,systemic anaphylaxis, systemic inflammatory response syndrome, systemiconset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia,thromboangitis obliterans, thrombocytopenia, toxicity, transplants,trauma/hemorrhage, type III hypersensitivity reactions, type IVhypersensitivity, unstable angina, uremia, urosepsis, urticaria,valvular heart diseases, varicose veins, vasculitis, venous diseases,venous thrombosis, ventricular fibrillation, viral and fungalinfections, vital encephalitis/aseptic meningitis, vital-associatedhemaphagocytic syndrome, Wernicke-Korsakoff syndrome, Wilson's disease,xenograft rejection of any organ or tissue, acute coronary syndromes,acute idiopathic polyneuritis, acute inflammatory demyelinatingpolyradiculoneuropathy, acute ischemia, adult Still's disease,anaphylaxis, anti-phospholipid antibody syndrome, aplastic anemia,atopic eczema, atopic dermatitis, autoimmune dermatitis, autoimmunedisorder associated with streptococcus infection, autoimmuneenteropathy, autoimmune hearing loss, autoimmune lymphoproliferativesyndrome (ALPS), autoimmune myocarditis, autoimmune premature ovarianfailure, blepharitis, bronchiectasis, bullous pemphigoid, cardiovasculardisease, catastrophic antiphospholipid syndrome, celiac disease,cervical spondylosis, chronic ischemia, cicatricial pemphigoid,clinically isolated syndrome (cis) with risk for multiple sclerosis,childhood onset psychiatric disorder, dacryocystitis, dermatomyositis,diabetic retinopathy, disk herniation, disk prolapse, drug inducedimmune hemolytic anemia, endometriosis, endophthalmitis, episcleritis,erythema multiforme, erythema multiforme major, gestational pemphigoid,Guillain-Barr-syndrome (GBS), hay fever, Hughes syndrome, idiopathicParkinson's disease, idiopathic interstitial pneumonia, IgE-mediatedallergy, immune hemolytic anemia, inclusion body myositis, infectiousocular inflammatory disease, inflammatory demyelinating disease,inflammatory heart disease, inflammatory kidney disease, IPF/UIP,iritis, keratitis, keratoconjunctivitis sicca, Kussmaul disease orKussmaul-Meier disease, Landry's paralysis, Langerhan's cellhistiocytosis, livedo reticularis, macular degeneration, microscopicpolyangiitis, morbus bechterev, motor neuron disorders, mucous membranepemphigoid, multiple organ failure, myelodysplastic syndrome,myocarditis, nerve root disorders, neuropathy, non-A non-B hepatitis,optic neuritis, osteolysis, ovarian cancer, pauciarticular JRA,peripheral artery occlusive disease (PAOD), peripheral vascular disease(PVD), peripheral artery, disease (PAD), phlebitis, polyarteritis nodosa(or periarteritis nodosa), polychondritis, polymyalgia rheumatica,poliosis, polyarticular JRA, polyendocrine deficiency syndrome,polymyositis, post-pump syndrome, primary Parkinsonism, prostate andrectal cancer and hematopoietic malignancies (leukemia and lymphoma),prostatitis, pure red cell aplasia, primary adrenal insufficiency,recurrent neuromyelitis optica, restenosis, rheumatic heart disease,sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis),scleroderma, secondary amyloidosis, shock lung, scleritis, sciatica,secondary adrenal insufficiency, silicone associated connective tissuedisease, Sneddon-Wilkinson dermatosis, spondilitis ankylosans,Stevens-Johnson syndrome (SJS), systemic inflammatory response syndrome,temporal arteritis, toxoplasmic retinitis, toxic epidermal necrolysis,transverse myelitis, TRAPS (tumor necrosis factor receptor-associatedperiodic syndrome), type 1 allergic reaction, type II diabetes, usualinterstitial pneumonia (UIP), vernal conjunctivitis, viral retinitis,Vogt-Koyanagi-Harada syndrome (VKH syndrome), wet macular degeneration,and wound healing.
 48. The method according to claim 47, wherein thebinding protein is formulated for parenteral, subcutaneous,intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermaladministration.
 49. A method of determining the presence, amount, orconcentration of at least one target or fragment thereof in a testsample by an immunoassay, wherein the immunoassay comprises contactingthe test sample with at least one binding protein and at least onedetectable label; and wherein the at least one binding protein comprisesthe binding protein of claim
 26. 50. The method of claim 49, furthercomprising: (a) contacting the test sample with the at least one bindingprotein, wherein the binding protein binds to an epitope on the targetor fragment thereof so as to form a first complex; (b) contacting thecomplex with the at least one detectable label, wherein the detectablelabel binds to the binding protein or an epitope on the target orfragment thereof that is not bound by the binding protein to form asecond complex; and (c) detecting the presence, amount, or concentrationof the target or fragment thereof in the test sample based on the signalgenerated by the detectable label in the second complex, wherein thepresence, amount, or concentration of the target or fragment thereof isdirectly correlated with the signal generated by the detectable label.51. The method of claim 49, further comprising: (a) contacting the testsample with the at least one binding protein, wherein the bindingprotein binds to an epitope on the target or fragment thereof so as toform a first complex; (b) contacting the complex with the at least onedetectable label, wherein the detectable label competes with the targetor fragment thereof for binding to the binding protein so as to form asecond complex; and (c) detecting the presence, amount, or concentrationof the target or fragment thereof in the test sample based on the signalgenerated by the detectable label in the second complex, wherein thepresence, amount, or concentration of the target or fragment thereof isindirectly correlated with the signal generated by the detectable label.52. A kit for assaying a test sample for the presence, amount, orconcentration of a target or fragment thereof in the sample, said kitcomprising: (a) instructions for assaying the test sample for the targetor fragment thereof; and (b) at least one binding protein comprising thebinding protein of claim 26.